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Sample records for microbial proteases compromise

  1. Production of Microbial Protease from Selected Soil Fungal Isolates ...

    African Journals Online (AJOL)

    Production of Microbial Protease from Selected Soil Fungal Isolates. ... Nigerian Journal of Biotechnology ... and 500C. The optimal pH on the enzyme production was observed to be between pH 3.5 and 5.5 for the organisms. Keywords: Soil microorganism, fungal isolate, incubation period, microbial enzyme. Nig J. Biotech.

  2. Compromises.

    Science.gov (United States)

    Sizer, Theodore R.

    1984-01-01

    Taking as examples the issues of improving students'"high order thinking skills" and arriving at more equitable teacher salaries and school budgets, the author discusses the need for compromise solutions to widespread problems. (JBM)

  3. Microbial alkaline proteases: Optimization of production parameters and their properties

    Directory of Open Access Journals (Sweden)

    Kanupriya Miglani Sharma

    2017-06-01

    Full Text Available Proteases are hydrolytic enzymes capable of degrading proteins into small peptides and amino acids. They account for nearly 60% of the total industrial enzyme market. Proteases are extensively exploited commercially, in food, pharmaceutical, leather and detergent industry. Given their potential use, there has been renewed interest in the discovery of proteases with novel properties and a constant thrust to optimize the enzyme production. This review summarizes a fraction of the enormous reports available on various aspects of alkaline proteases. Diverse sources for isolation of alkaline protease producing microorganisms are reported. The various nutritional and environmental parameters affecting the production of alkaline proteases in submerged and solid state fermentation are described. The enzymatic and physicochemical properties of alkaline proteases from several microorganisms are discussed which can help to identify enzymes with high activity and stability over extreme pH and temperature, so that they can be developed for industrial applications.

  4. Anti-fibrinolytic and anti-microbial activities of a serine protease inhibitor from honeybee (Apis cerana) venom.

    Science.gov (United States)

    Yang, Jie; Lee, Kwang Sik; Kim, Bo Yeon; Choi, Yong Soo; Yoon, Hyung Joo; Jia, Jingming; Jin, Byung Rae

    2017-10-01

    Bee venom contains a variety of peptide constituents, including low-molecular-weight protease inhibitors. While the putative low-molecular-weight serine protease inhibitor Api m 6 containing a trypsin inhibitor-like cysteine-rich domain was identified from honeybee (Apis mellifera) venom, no anti-fibrinolytic or anti-microbial roles for this inhibitor have been elucidated. In this study, we identified an Asiatic honeybee (A. cerana) venom serine protease inhibitor (AcVSPI) that was shown to act as a microbial serine protease inhibitor and plasmin inhibitor. AcVSPI was found to consist of a trypsin inhibitor-like domain that displays ten cysteine residues. Interestingly, the AcVSPI peptide sequence exhibited high similarity to the putative low-molecular-weight serine protease inhibitor Api m 6, which suggests that AcVSPI is an allergen Api m 6-like peptide. Recombinant AcVSPI was expressed in baculovirus-infected insect cells, and it demonstrated inhibitory activity against trypsin, but not chymotrypsin. Additionally, AcVSPI has inhibitory effects against plasmin and microbial serine proteases; however, it does not have any detectable inhibitory effects on thrombin or elastase. Consistent with these inhibitory effects, AcVSPI inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products. AcVSPI also bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi as well as gram-positive and gram-negative bacteria. These findings demonstrate the anti-fibrinolytic and anti-microbial roles of AcVSPI as a serine protease inhibitor. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Microbial Proteases in Baked Goods: Modification of Gluten and Effects on Immunogenicity and Product Quality

    Directory of Open Access Journals (Sweden)

    Nina G. Heredia-Sandoval

    2016-08-01

    Full Text Available Gluten-related diseases are a range of inflammatory disorders of the small intestine, characterized by an adverse response to gluten ingestion; therefore, the treatment is a gluten withdrawal. In spite of the increased market of gluten-free products, widely available breads with high acceptability are still missing due to the technological challenge of substituting the special gluten properties. Instead of using alternative ingredients for baking, some attempts have been done to decrease gluten immunogenicity by its enzymatic degradation with microbial proteases. Although the gluten immunogenicity reduction has been reached to an acceptable level, some quality parameters of the products are affected. This review focus on the use of microbial peptidases to prepare less immunogenic baked goods and their effect on product quality.

  6. Microbial Proteases in Baked Goods: Modification of Gluten and Effects on Immunogenicity and Product Quality.

    Science.gov (United States)

    Heredia-Sandoval, Nina G; Valencia-Tapia, Maribel Y; Calderón de la Barca, Ana M; Islas-Rubio, Alma R

    2016-08-30

    Gluten-related diseases are a range of inflammatory disorders of the small intestine, characterized by an adverse response to gluten ingestion; therefore, the treatment is a gluten withdrawal. In spite of the increased market of gluten-free products, widely available breads with high acceptability are still missing due to the technological challenge of substituting the special gluten properties. Instead of using alternative ingredients for baking, some attempts have been done to decrease gluten immunogenicity by its enzymatic degradation with microbial proteases. Although the gluten immunogenicity reduction has been reached to an acceptable level, some quality parameters of the products are affected. This review focus on the use of microbial peptidases to prepare less immunogenic baked goods and their effect on product quality.

  7. Antibodies against homologous microbial caseinolytic proteases P characterise primary biliary cirrhosis.

    Science.gov (United States)

    Bogdanos, Dimitrios-Petrou; Baum, Harold; Sharma, Umesh C; Grasso, Alessandro; Ma, Yun; Burroughs, Andrew K; Vergani, Diego

    2002-01-01

    Antibodies to caseinolytic protease P(177-194) (ClpP(177-194)) of the proteolytic subunit of the Clp complex of Escherichia coli (E. coli) are uniquely present in primary biliary cirrhosis (PBC). Molecular mimicry between the regulatory subunit ClpX and the principal T-cell epitope of pyruvate dehydrogenase complex (PDC-E2) in PBC, has been proposed to account for this. Since ClpP is highly conserved among bacteria we investigated whether the micro-organisms triggering these antibodies may be other than E. coli. E. coli ClpP(177-194) is homologous with ClpP peptides of Yersinia enterocolitica (YEREN) and Haemophilus influenzae (HAEIN). Enzyme linked immunosorbent assay (ELISA) reactivity to these peptides was tested in 45 patients with PBC, 44 pathological and 32 healthy controls. Reactivity to at least one of the ClpP peptides was observed in 21 (47%) PBC patients, 5.8% pathological and 3.1% healthy controls (PECOLI ClpP(177-194), alone or in association with YEREN and/or HAEIN peptides, compared to three (14.2%) reactive with YEREN, two (9.5%) with YEREN/HAEIN and one (4.7%) with HAEIN peptide. Simultaneous reactivity to homologous sequences was due to cross-reactivity as confirmed by competition ELISAs. The PBC-specificity of anti-microbial ClpP reactivity is confirmed: the questions as to primary trigger(s) and relevance to PBC pathogenesis remain open.

  8. Detergent-compatible proteases: microbial production, properties, and stain removal analysis.

    Science.gov (United States)

    Niyonzima, Francois Niyongabo; More, Sunil

    2015-01-01

    Proteases are one of the most important commercial enzymes used in various industrial domains such as detergent and leather industries. The alkaline proteases as well as other detergent-compatible enzymes such as lipases and amylases serve now as the key components in detergent formulations. They break down various stains during fabric washing. The search for detergent-compatible proteases with better properties is a continuous exercise. The current trend is to use detergent-compatible proteases that are stable over a wide temperature range. Although the proteases showing stability at elevated pH have the capacity to be used in detergent formulations, their usage can be significant if they are also stable and compatible with detergent and detergent ingredients, and also able to remove protein stains. Despite the existence of some reviews on alkaline proteases, there is no specification for the use of alkaline proteases as detergent additives. The present review describes the detergent-compatible proteases tested as detergent additives. An overview was provided for screening, optimization, purification, and properties of detergent compatible proteases, with an emphasis on the stability and compatibility of the alkaline proteases with the detergent and detergent compounds, as well as stain removal examination methods.

  9. Loss of second and sixth conserved cysteine residues from trypsin inhibitor-like cysteine-rich domain-type protease inhibitors in Bombyx mori may induce activity against microbial proteases.

    Science.gov (United States)

    Li, Youshan; Liu, Huawei; Zhu, Rui; Xia, Qingyou; Zhao, Ping

    2016-12-01

    Previous studies have indicated that most trypsin inhibitor-like cysteine-rich domain (TIL)-type protease inhibitors, which contain a single TIL domain with ten conserved cysteines, inhibit cathepsin, trypsin, chymotrypsin, or elastase. Our recent findings suggest that Cys 2nd and Cys 6th were lost from the TIL domain of the fungal-resistance factors in Bombyx mori, BmSPI38 and BmSPI39, which inhibit microbial proteases and the germination of Beauveria bassiana conidia. To reveal the significance of these two missing cysteines in relation to the structure and function of TIL-type protease inhibitors in B. mori, cysteines were introduced at these two positions (D36 and L56 in BmSPI38, D38 and L58 in BmSPI39) by site-directed mutagenesis. The homology structure model of TIL domain of the wild-type and mutated form of BmSPI39 showed that two cysteine mutations may cause incorrect disulfide bond formation of B. mori TIL-type protease inhibitors. The results of Far-UV circular dichroism (CD) spectra indicated that both the wild-type and mutated form of BmSPI39 harbored predominantly random coil structures, and had slightly different secondary structure compositions. SDS-PAGE and Western blotting analysis showed that cysteine mutations affected the multimerization states and electrophoretic mobility of BmSPI38 and BmSPI39. Activity staining and protease inhibition assays showed that the introduction of cysteine mutations dramaticly reduced the activity of inhibitors against microbial proteases, such as subtilisin A from Bacillus licheniformis, protease K from Engyodontium album, protease from Aspergillus melleus. We also systematically analyzed the key residue sites, which may greatly influence the specificity and potency of TIL-type protease inhibitors. We found that the two missing cysteines in B. mori TIL-type protease inhibitors might be crucial for their inhibitory activities against microbial proteases. The genetic engineering of TIL-type protease inhibitors may be

  10. Towards generation of bioactive peptides from meat industry waste proteins: Generation of peptides using commercial microbial proteases.

    Science.gov (United States)

    Ryder, Kate; Bekhit, Alaa El-Din; McConnell, Michelle; Carne, Alan

    2016-10-01

    Five commercially available food-grade microbial protease preparations were evaluated for their ability to hydrolyse meat myofibrillar and connective tissue protein extracts to produce bioactive peptides. A bacterial-derived protease (HT) extensively hydrolysed both meat protein extracts, producing peptide hydrolysates with significant in vitro antioxidant and ACE inhibitor activities. The hydrolysates retained bioactivity after simulated gastrointestinal hydrolysis challenge. Gel permeation chromatography sub-fractionation of the crude protein hydrolysates showed that the smaller peptide fractions exhibited the highest antioxidant and ACE inhibitor activities. OFFGEL electrophoresis of the small peptides of both hydrolysates showed that low isoelectric point peptides had antioxidant activity; however, no consistent relationship was observed between isoelectric point and ACE inhibition. Cell-based assays indicated that the hydrolysates present no significant cytotoxicity towards Vero cells. The results indicate that HT protease hydrolysis of meat myofibrillar and connective tissue protein extracts produces bioactive peptides that are non-cytotoxic, should be stable in the gastrointestinal tract and may contain novel bioactive peptide sequences. Copyright © 2016 Elsevier Ltd. All rights reserved.

  11. Protease inhibitor associated mutations compromise the efficacy of therapy in human immunodeficiency virus – 1 (HIV-1 infected pediatric patients: a cross-sectional study

    Directory of Open Access Journals (Sweden)

    Petrova Anna

    2007-07-01

    Full Text Available Abstract Background Although the introduction of combined therapy with reverse transcriptase and protease inhibitors has resulted in considerable decrease in HIV related mortality; it has also induced the development of multiple drug-resistant HIV-1 variants. The few studies on HIV-1 mutagenesis in HIV infected children have not evaluated the impact of HIV-1 mutations on the clinical, virological and immunological presentation of HIV disease that is fundamental to optimizing the treatment regimens for these patients. Results A cross sectional study was conducted to evaluate the impact of treatment regimens and resistance mutation patterns on the clinical, virological, and immunological presentation of HIV disease in 41 children (25 male and 16 female at the Robert Wood Johnson Pediatric AIDS Program in New Brunswick, New Jersey. The study participants were symptomatic and had preceding treatment history with combined ARV regimens including protease inhibitors (PIs, nucleoside reverse transcriptase inhibitors (NRTIs and non-nucleoside reverse transcriptase inhibitors (NNRTIs. Fifteen (36.6% children were treated with NRTI+NNRTI+ PI, 6 (14.6% with NRTI+NNRTIs, 13 (31.7% with NRTI+PIs, and the remaining 7 (17.1% received NRTIs only. Combined ARV regimens did not significantly influence the incidence of NRTI and NNRTI associated mutations. The duration of ARV therapy and the child's age had no significant impact on the ARV related mutations. The clinico-immunological presentation of the HIV disease was not associated with ARV treatment regimens or number of resistance mutations. However, primary mutations in the protease (PR gene increased the likelihood of plasma viral load (PVL ≥ 10,000 copies/mL irrespective of the child's age, duration of ARV therapy, presence of NRTI and NNRTI mutation. Viremia ≥ 10,000 copies/mL was recorded in almost all the children with primary mutations in the PR region (n = 12/13, 92.3% as compared with only 50.0% (n

  12. [Comparative characteristics of microbial proteases by the level of hydrolysis of protein substrates].

    Science.gov (United States)

    Rimareva, L V; Overchenko, M B; Serba, E M; Trifonova, V V

    1997-01-01

    Screening of enzyme preparations displaying a maximum proteolytic activity at pH 4.0-5.5 and effecting deep proteolysis of plant proteins was performed. Amyloprotooryzin prepared from Aspergillus oryzae 387 containing a complex of proteolytic enzymes was the most effective. The amino acid composition of the hydrolysates obtained was studied. Amyloprotooryzin increased the contents of amino acids by 108-227%, depending on the substrate used. The enzymatic complex of amyloprotooryzin was studied; in addition, proteases, alpha-amylase, exo-beta-glucanase, and xylanase were detected in the complex.

  13. Protease inhibitors as a possible new factor in agricultural plant protection against microbial and fungal attack

    Czech Academy of Sciences Publication Activity Database

    Navrátil, Oldřich; Kodrík, Dalibor; Kludkiewicz, Barbara; Vinokurov, Konstantin; Sehnal, František; Horáčková, V.

    2012-01-01

    Roč. 73, Jan 2012 (2012), s. 61-67 ISSN 1027-3115 R&D Projects: GA MŠk 1M06030; GA MZe QI91A229 Institutional research plan: CEZ:AV0Z50380511; CEZ:AV0Z50070508 Keywords : potato * transgenosis * microbial and fungal pathogens Subject RIV: EB - Genetics ; Molecular Biology http://home.ueb.cas.cz/publikace/2012_Navratil_IOBC_61-67.pdf

  14. Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.

    Science.gov (United States)

    Clancy, Danielle M; Sullivan, Graeme P; Moran, Hannah B T; Henry, Conor M; Reeves, Emer P; McElvaney, Noel G; Lavelle, Ed C; Martin, Seamus J

    2018-03-13

    Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  15. Nonadhesive, silica nanoparticles-based brush-coated contact lens cases--compromising between ease of cleaning and microbial transmission to contact lenses.

    Science.gov (United States)

    Qu, Wenwen; Hooymans, Johanna M M; Qiu, Jun; de-Bont, Nik; Gelling, Onko-Jan; van der Mei, Henny C; Busscher, Henk J

    2013-05-01

    Surface properties of lens cases are determinant for their cleanability and for microbial transmission from lens cases to contact lenses (CLs). PEG-polymer-brush-coatings are known to decrease microbial adhesion more than other surface-coatings. Here, we applied a robust, silica nanoparticles-based brush-coating to polypropylene cases to evaluate their ease of cleaning and probability of bacterial transmission to CLs. Adhesion forces of nine bacterial strains (Pseudomonas, Staphylococci, and Serratia) to rigid CLs, polypropylene, and silica nanoparticles-based brush-coated polypropylene were measured using atomic-force-microscopy and subjected to Weibull analyses to yield bacterial transmission probabilities. Biofilms of each strain were grown in coated and uncoated cases and rinsed with a NaCl or antimicrobial lens care solution. Residual, viable organisms were quantified. Bacterial adhesion forces of all strains were significantly, up to tenfold smaller on brush-coated than on uncoated polypropylene. This yielded, higher transmission probabilities to a CL, but mild-rinsing yielded 10-100 fold higher removal of bacteria from brush-coated than from polypropylene cases. Moreover, due to weak adhesion forces, bacteria on brush-coated cases were two-to-three fold more susceptible to an antimicrobial lens care solution than on polypropylene cases. Therewith, the design of lens case surfaces is a compromise between ease of cleaning and transmission probability to CLs. Copyright © 2013 Wiley Periodicals, Inc.

  16. Strained Compromises?

    DEFF Research Database (Denmark)

    Ibsen, Christian Lyhne

    The Danish version of flexicurity is not only about a balance between labour market flexibility and social security. Arguably, it is also series of more or less stable underlying compromises between social partners about the main mechanisms and aims of labour market regulation which – supposedly...... – should be focused on employment rather than jobs and competition on quality rather than on labour costs. However, most studies on Danish flexicurity have been carried out under favourable economic conditions with social partners almost naturally agreeing to the merits of the model – at least in principle...

  17. Nonadhesive, silica nanoparticles-based brush-coated contact lens casesCompromising between ease of cleaning and microbial transmission to contact lenses

    NARCIS (Netherlands)

    Qu, Wenwen; Hooymans, Johanna M. M.; Qiu, Jun; de-Bont, Nik; Gelling, Onko-Jan; van der Mei, Henny C.; Busscher, Henk J.

    Surface properties of lens cases are determinant for their cleanability and for microbial transmission from lens cases to contact lenses (CLs). PEG-polymer-brush-coatings are known to decrease microbial adhesion more than other surface-coatings. Here, we applied a robust, silica nanoparticles-based

  18. Microbial transformation of vanillin isolated from melia azedarach to vanillyl alcohol followed by protease inhibition and antioxidant activity

    International Nuclear Information System (INIS)

    Shahwar, D.; Raza, M.A.; Ali, T.; Ahmad, V.U.

    2011-01-01

    Vanillin [1) was isolated from ethanolic extract of Melia azedarach L. and incubated in the culture of Aspergillus niger GC-4, which resulted in vanillyl alcohol (2). The product was identified by mass, /sup 1/H and 13C-NMR spectroscopy. Protease inhibition potential was measured using in vitro model, which indicated significant enzyme inhibition activity of vanillyl alcohol as compared to vanillin (1). Compound 1 and 2 were screened for antioxidant activity using phosphomolybdate and diphenyl picrylhydrazyl (DPPH) in vitro model systems. Dose-dependant increase in the antioxidant activity of vanillyl alcohol was observed in phosphomolybdate method, while vanillin was found to be inactive. Vanillyl alcohol showed remarkable decay of the free radicals in the solution of DPPH within first two minutes which finally became constant after five minutes. In contrast, vanillin exhibited much lower antioxidant activity. (author)

  19. Supermarket Proteases.

    Science.gov (United States)

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

    Presents a laboratory activity on enzymes. Uses common items found in the supermarket that contain protease enzymes, such as contact lens cleaner and meat tenderizer. Demonstrates the digestion of gelatin proteins as part of enzymatic reactions. (Author/SOE)

  20. Earthworm Protease

    Directory of Open Access Journals (Sweden)

    Rong Pan

    2010-01-01

    Full Text Available The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibriniolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP. The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate proenzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  1. Earthworm Protease

    International Nuclear Information System (INIS)

    Pan, R.; Zhang, Z.; He, R.

    2010-01-01

    The alimentary tract of earthworm secretes a group of proteases with a relative wide substrate specificity. In 1983, six isozymes were isolated from earthworm with fibrinolytic activities and called fibrinolytic enzymes. So far, more isozymes have been found from different earthworm species such as Lumbricus rubellus and Eisenia fetida. For convenience, the proteases are named on the basis of the earthworm species and the protein function, for instance, Eisenia fetida protease (EfP). The proteases have the abilities not only to hydrolyze fibrin and other protein, but also activate pro enzymes such as plasminogen and prothrombin. In the light of recent studies, eight of the EfPs contain oligosaccharides chains which are thought to support the enzyme structure. Interestingly, EfP-II has a broader substrate specificity presenting alkaline trypsin, chymotrypsin and elastase activities, but EfP-III-1 has a stricter specificity. The protein crystal structures show the characteristics in their specificities. Earthworm proteases have been applied in several areas such as clinical treatment of clotting diseases, anti-tumor study, environmental protection and nutritional production. The current clinical utilizations and some potential new applications of the earthworm protease will be discussed in this paper.

  2. Democratic Respect and Compromise

    DEFF Research Database (Denmark)

    Rostbøll, Christian F.

    2017-01-01

    Compromise has attained renewed interest among political theorists writing on pluralism and disagreement. It is controversial, however, whether compromise is a mere pragmatic necessity or if it has non-instrumental value. This article argues that the reasons for compromise are inherent in the dem......Compromise has attained renewed interest among political theorists writing on pluralism and disagreement. It is controversial, however, whether compromise is a mere pragmatic necessity or if it has non-instrumental value. This article argues that the reasons for compromise are inherent...

  3. High throughput in vivo protease inhibitor selection platform

    DEFF Research Database (Denmark)

    2017-01-01

    The invention relates to a recombinant microbial cell comprising a selection platform for screening for a protease inhibitor, wherein the platform comprises transgenes encoding a protease having selective peptide bond cleavage activity at a recognition site amino acid sequence; and transgenes...... platform for screening for a protease inhibitor....

  4. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    -terminal of the scissile bond, leaving C-terminal fusions to have non-native C-termini after processing. A solution yielding native C-termini would allow novel expression and purification systems for therapeutic proteins and peptides.The peptidyl-Lys metallopeptidase (LysN) of the fungus Armillaria mellea (Am) is one...... of few known proteases to have substrate specificity for the C-terminal side of the scissile bond. LysN exhibits specificity for lysine, and has primarily been used to complement trypsin in to proteomic studies. A working hypothesis during this study was the potential of LysN as a processing protease...

  5. Supplementation of broiler diets with high levels of microbial protease and phytase enables partial replacement of commercial soybean meal with raw, full-fat soybean.

    Science.gov (United States)

    Erdaw, M M; Perez-Maldonado, R A; Iji, P A

    2018-02-22

    A 3 × 3 + 1 factorial, involving three levels of protease (0, 15,000 or 30,000 PROT/kg) and three levels of phytase (1,000, 2,000 or 3,000 FYT/kg), was used to evaluate the effect of replacing commercial soybean meal (SBM) with raw, full-fat soybean (RFSB) at 75 g/kg of diet for broilers. A control diet was used for comparison. Each treatment was replicated six times, with nine birds per replicate. The concentration of trypsin inhibitors (TIs) in the test diets was approximately 10,193.4 TIU/kg. Regardless of enzyme supplementation, feed intake (FI) and body weight gain (BWG) of birds in the control group were superior to those on the test diets. Birds that received the protease-free test diets had reduced FI and BWG, but when supplemented with protease, were similar to the control diet in BWG, FI (except 0-35 days) and feed conversion ratio (FCR). When the test diet was supplemented with elevated levels (extradose) of protease and phytase, the BWG was improved during 0-10 days (p = .05) and 0-24 days (p replace SBM in broiler diets, provided the diets are supplemented with elevated levels of protease and phytase. © 2018 Blackwell Verlag GmbH.

  6. Compromise and Toleration

    DEFF Research Database (Denmark)

    Rostbøll, Christian F.

    Political compromise is akin to toleration, since both consist of an "agreement to disagree." Compromise and toleration also share a predicament of being regarded as ambiguous virtues that require of us to accept something we actually regard as wrong. However, we misunderstand the nature, justifi...... in compromise are more stringent than those for being tolerated. Still, the limits of compromise cannot be drawn to narrowly if it is to remain its value as a form of agreement that respects and embodies the differences of opinion in society.......Political compromise is akin to toleration, since both consist of an "agreement to disagree." Compromise and toleration also share a predicament of being regarded as ambiguous virtues that require of us to accept something we actually regard as wrong. However, we misunderstand the nature......, justification, and limits of compromise if we see it merely as a matter of toleration. While toleration is mainly a matter of accepting citizens' equal right to co-existence as subjects to law, political compromise includes the parties in making law – it makes them co-authors of law. Toleration entails...

  7. [Tuberculosis in compromised hosts].

    Science.gov (United States)

    2003-11-01

    Recent development of tuberculosis in Japan tends to converge on a specific high risk group. The proportion of tuberculosis developing particularly from the compromised hosts in the high risk group is especially high. At this symposium, therefore, we took up diabetes mellitus, gastrectomy, dialysis, AIDS and the elderly for discussion. Many new findings and useful reports for practical medical treatment are submitted; why these compromised hosts are predisposed to tuberculosis, tuberculosis diagnostic and remedial notes of those compromised hosts etc. It is an important question for the future to study how to prevent tuberculosis from these compromised hosts. 1. Tuberculosis in diabetes mellitus: aggravation and its immunological mechanism: Kazuyoshi KAWAKAMI (Department of Internal Medicine, Division of Infectious Diseases, Graduate School and Faculty of Medicine, University of the Ryukyus). It has been well documented that diabetes mellitus (DM) is a major aggravating factor in tuberculosis. The onset of this disease is more frequent in DM patients than in individuals with any underlying diseases. However, the precise mechanism of this finding remains to be fully understood. Earlier studies reported that the migration, phagocytosis and bactericidal activity of neutrophils are all impaired in DM patients, which is related to their reduced host defense to infection with extracellular bacteria, such as S. aureus and E. colli. Host defense to mycobacterial infection is largely mediated by cellular immunity, and Th1-related cytokines, such as IFN-gamma and IL-12, play a central role in this response. It is reported that serum level of these cytokines and their production by peripheral blood mononuclear cells (PBMC) are reduced in tuberculosis patients with DM, and this is supposed to be involved in the high incidence of tuberculosis in DM. Our study observed similar findings and furthermore indicated that IFN-gamma and IL-12 production by BCG-stimulated PBMC was lower

  8. A biotechnology perspective of fungal proteases

    Directory of Open Access Journals (Sweden)

    Paula Monteiro de Souza

    2015-06-01

    Full Text Available Proteases hydrolyze the peptide bonds of proteins into peptides and amino acids, being found in all living organisms, and are essential for cell growth and differentiation. Proteolytic enzymes have potential application in a wide number of industrial processes such as food, laundry detergent and pharmaceutical. Proteases from microbial sources have dominated applications in industrial sectors. Fungal proteases are used for hydrolyzing protein and other components of soy beans and wheat in soy sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications.

  9. Effects of Protease, Phytase and a Bacillus sp. Direct-Fed Microbial on Nutrient and Energy Digestibility, Ileal Brush Border Digestive Enzyme Activity and Cecal Short-Chain Fatty Acid Concentration in Broiler Chickens

    Science.gov (United States)

    Murugesan, Ganapathi R.; Romero, Luis F.; Persia, Michael E.

    2014-01-01

    Two experiments were conducted to determine the effects of protease and phytase (PP) and a Bacillus sp. direct-fed microbial (DFM) on dietary energy and nutrient utilization in broiler chickens. In the first experiment, Ross 308 broiler chicks were fed diets supplemented with PP and DFM in a 2×2 factorial arrangement. The 4 diets (control (CON), CON + PP, CON + DFM, and CON + PP + DFM) were fed from 15–21 days of age. In Experiment 1, significant interaction (P≤0.01) between PP and DFM on the apparent ileal digestibility coefficient for starch, crude protein, and amino acid indicated that both additives increased the digestibility. Both additives increased the nitrogen retention coefficient with a significant interaction (P≤0.01). Although no interaction was observed, significant main effects (P≤0.01) for nitrogen-corrected apparent ME (AMEn) for PP or DFM indicated an additive response. In a follow-up experiment, Ross 308 broiler chicks were fed the same experimental diets from 1–21 days of age. Activities of ileal brush border maltase, sucrase, and L-alanine aminopeptidase were increased (P≤0.01) by PP addition, while a trend (P = 0.07) for increased sucrase activity was observed in chickens fed DFM, in Experiment 2. The proportion of cecal butyrate was increased (P≤0.01) by DFM addition. Increased nutrient utilization and nitrogen retention appear to involve separate but complementary mechanisms for PP and DFM, however AMEn responses appear to have separate and additive mechanisms. PMID:25013936

  10. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    signalling to short-circuit host cell processes. Common to both intra- and extracellular proteases is the tight control of their proteolytic activities. In general, substrate recognition by the intracellular proteases is highly selective which is, in part, attributed to the chaperone activity associated...... tolerance to adverse conditions such as those experienced in the host. In the membrane, HtrA performs similar functions whereas the extracellular proteases, in close contact with host components, pave the way for spreading infections by degrading host matrix components or interfering with host cell...... with the proteases either encoded within the same polypeptide or on separate subunits. In contrast, substrate recognition by extracellular proteases is less selective and therefore these enzymes are generally expressed as zymogens to prevent premature proteolytic activity that would be detrimental to the cell...

  11. Dysregulation of protease and protease inhibitors in a mouse model of human pelvic organ prolapse.

    Directory of Open Access Journals (Sweden)

    Madhusudhan Budatha

    Full Text Available Mice deficient for the fibulin-5 gene (Fbln5(-/- develop pelvic organ prolapse (POP due to compromised elastic fibers and upregulation of matrix metalloprotease (MMP-9. Here, we used casein zymography, inhibitor profiling, affinity pull-down, and mass spectrometry to discover additional protease upregulated in the vaginal wall of Fbln5(-/- mice, herein named V1 (25 kDa. V1 was a serine protease with trypsin-like activity similar to protease, serine (PRSS 3, a major extrapancreatic trypsinogen, was optimum at pH 8.0, and predominantly detected in estrogenized vaginal epithelium of Fbln5(-/- mice. PRSS3 was (a localized in epithelial secretions, (b detected in media of vaginal organ culture from both Fbln5(-/- and wild type mice, and (c cleaved fibulin-5 in vitro. Expression of two serine protease inhibitors [Serpina1a (α1-antitrypsin and Elafin] was dysregulated in Fbln5(-/- epithelium. Finally, we confirmed that PRSS3 was expressed in human vaginal epithelium and that SERPINA1 and Elafin were downregulated in vaginal tissues from women with POP. These data collectively suggest that the balance between proteases and their inhibitors contributes to support of the pelvic organs in humans and mice.

  12. Construction and application of a novel genetically engineered Aspergillus oryzae for expressing proteases

    Directory of Open Access Journals (Sweden)

    Xiao-Chun Yu

    2017-09-01

    Conclusions: These findings suggest that cloning of microbial protease genes with good physicochemical properties and expressing them in an ideal host such as A. oryzae is a novel strategy to enhance the value of soybean meal.

  13. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

    Science.gov (United States)

    Selzer, Paul M.; Pingel, Sabine; Hsieh, Ivy; Ugele, Bernhard; Chan, Victor J.; Engel, Juan C.; Bogyo, Matthew; Russell, David G.; Sakanari, Judy A.; McKerrow, James H.

    1999-09-01

    Papain family cysteine proteases are key factors in the pathogenesis of cancer invasion, arthritis, osteoporosis, and microbial infections. Targeting this enzyme family is therefore one strategy in the development of new chemotherapy for a number of diseases. Little is known, however, about the efficacy, selectivity, and safety of cysteine protease inhibitors in cell culture or in vivo. We now report that specific cysteine protease inhibitors kill Leishmania parasites in vitro, at concentrations that do not overtly affect mammalian host cells. Inhibition of Leishmania cysteine protease activity was accompanied by defects in the parasite's lysosome/endosome compartment resembling those seen in lysosomal storage diseases. Colocalization of anti-protease antibodies with biotinylated surface proteins and accumulation of undigested debris and protease in the flagellar pocket of treated parasites were consistent with a pathway of protease trafficking from flagellar pocket to the lysosome/endosome compartment. The inhibitors were sufficiently absorbed and stable in vivo to ameliorate the pathology associated with a mouse model of Leishmania infection.

  14. Increasing the alkaline protease activity of Bacillus cereus and ...

    African Journals Online (AJOL)

    User

    2011-05-09

    May 9, 2011 ... cereus and Bacillus polymyxa simultaneously with the start of sporulation phase as a ... microbial forms to inactivation by chemical or physical agents. .... alkaline pH, 9, 10 and 11 and the pH of the culture media was optimized with .... incubation temperature for alkaline protease production by Bacillus ...

  15. Energy policy - compromise or change?

    International Nuclear Information System (INIS)

    Lewitz, J.C.

    1998-01-01

    Is there a contrast between compromise and change? In the author's opinion, there is not. Society changes. Sometimes, change is brought about more easily on the basis of a compromise. A pluralistic society needs compromises in order to participate in change with convictions of its own. To reach a compromise including a large part of one's conviction, the public must be convinced. This is achieved most easily by somebody who is well informed, educated, and trained. In this respect, not only specialized knowledge counts, but also the ability to handle the language and to know how to speak, and the behavior in public. Expert knowledge is acquired at the university, in the execution of one's profession, and in advanced training. Knowledge may be enhanced, for instance, by dealing with the arguments used by the other side. This will help in arguing one's own point more effectively. Individual talks, and events at which information and knowledge are disseminated, enable persons to assume an opinion based on their comprehension. Many uniform opinions create a majority opinion. (orig.) [de

  16. Flow-based Compromise Detection

    NARCIS (Netherlands)

    Hofstede, R.J.

    2016-01-01

    Brute-force attacks are omnipresent and manyfold on the Internet, and aim at compromising user accounts by issuing large numbers of authentication attempts on applications and daemons. Widespread targets of such attacks are Secure SHell (SSH) and Web applications, for example. The impact of

  17. Respiratory Failure Associated with the Lipodystrophy Syndrome in an HIV-Positive Patient with Compromised Lung Function

    Directory of Open Access Journals (Sweden)

    Natasha Press

    2001-01-01

    Full Text Available Protease inhibitors, used as treatment in human immunodeficiency virus (HIV infection, are associated with a syndrome of peripheral lipodystrophy, central adiposity, hyperlipidemia and insulin resistance. An HIV-positive patient with chronic obstructive pulmonary disease is presented who developed the lipodystrophy syndrome that is associated with the use of protease inhibitors. It is postulated that the lipodystrophy syndrome further compromised his lung function, leading to respiratory failure. Patients who have pulmonary disease and are taking protease inhibitors require monitoring of clinical status and pulmonary function tests.

  18. Proteases and antiproteases in chronic neutrophilic lung disease - relevance to drug discovery.

    LENUS (Irish Health Repository)

    Greene, Catherine M

    2009-10-01

    Chronic inflammatory lung diseases such as cystic fibrosis and emphysema are characterized by higher-than-normal levels of pulmonary proteases. While these enzymes play important roles such as bacterial killing, their dysregulated expression or activity can adversely impact on the inflammatory process. The existence of efficient endogenous control mechanisms that can dampen or halt this overexuberant protease activity in vivo is essential for the effective resolution of inflammatory lung disease. The function of pulmonary antiproteases is to fulfil this role. Interestingly, in addition to their antiprotease activity, protease inhibitors in the lung also often possess other intrinsic properties that contribute to microbial killing or termination of the inflammatory process. This review will outline important features of chronic inflammation that are regulated by pulmonary proteases and will describe the various mechanisms by which antiproteases attempt to counterbalance exaggerated protease-mediated inflammatory events. These proteases, antiproteases and their modifiers represent interesting targets for therapeutic intervention.

  19. Compromise and Disagreement in Comtemporary Political Theory

    DEFF Research Database (Denmark)

    Rostbøll, Christian F.; Scavenius, Theresa

    Compromise and Disagreement in Contemporary Political Theory provides a critical discussion of when and to what extent compromise is the best response to pluralism and disagreement in democratic decision-making and beyond. Organized into four parts, the book begins by discussing the justification...... and limits of compromise. Part II discusses the practice of compromise and considers the ethics required for compromise as well as the institutions that facilitate compromise. Part III focuses on pluralism and connects the topic of compromise to current discussions in political theory on public reason...

  20. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø

    2011-01-01

    , directed against blood coagulation factors, are in clinical trials as anticoagulant drugs. Several of the studies on protease-binding aptamers have been pioneering and trend-setting in the field. The work with protease-binding aptamers also demonstrates many interesting examples of non-standard selection......Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing...... small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of aptamer selection, proteinaptamer recognition, protease inhibition, and advantages of aptamers...

  1. The battle in the apoplast: further insights into the roles of proteases and their inhibitors in plant-pathogen interactions

    Directory of Open Access Journals (Sweden)

    Mansoor eKarimi Jashni

    2015-08-01

    Full Text Available Upon host penetration, fungal pathogens secrete a plethora of effectors to promote disease, including proteases that degrade plant antimicrobial proteins, and protease inhibitors (PIs that inhibit plant proteases with antimicrobial activity. Conversely, plants secrete proteases and PIs to protect themselves against pathogens or to mediate recognition of pathogen proteases and PIs, which leads to induction of defense responses. Many examples of proteases and PIs mediating effector-triggered immunity in host plants have been reported in the literature, but little is known about their role in compromising basal defense responses induced by microbe-associated molecular patterns. Recently, several reports appeared in literature on secreted fungal proteases that modify or degrade pathogenesis-related proteins, including plant chitinases or PIs that compromise their activities. This prompted us to review the recent advances on proteases and PIs involved in fungal virulence and plant defense. Proteases and PIs from plants and their fungal pathogens play an important role in the arms race between plants and pathogens, which has resulted in co-evolutionary diversification and adaptation shaping pathogen lifestyles.

  2. COMPROMISE EFFECT ON CONSUMERS’ BEHAVIOR

    Directory of Open Access Journals (Sweden)

    Markus Surkamta Eric Santosa

    2016-09-01

    Full Text Available The way consumers think about the products they will buy determines their buying behavior. The decision to buy a particular product is obviously in accordance with the buyer’s attitude. The buyers will also feel more comfortable if their behavior meets with the approval and expectations of the people close to them. While the development of a certain attitude has no effect on subjective judgment, the effect of compromise is likely to make a contribution to its development. Since it is still unclear, this study’s main purposed is to clarify this. In addition, while an attitude is theorized as being a predictor of behavior, through behavioral intention, the study’s secondary purpose is to boost the earlier findings. Likewise, in accordance with the theory of planned behavior, the study will also examine the other predictors of behavioral intention, in relation to the behavioral intention itself. A sample consisting of a 100 respondents was compiled by using the convenience and judgment technique. The data were analyzed using Amos 16.0 and SPSS 16.0. As expected, the compromise effect had a significant influence on whether the customers’ attitude or the subjective norm was the main determinant. Likewise, the customers’ attitude, the subjective norm and perceived behavioral control were confirmed as good predictors of customers’ behavioral intentions.

  3. Multifunctional Mitochondrial AAA Proteases.

    Science.gov (United States)

    Glynn, Steven E

    2017-01-01

    Mitochondria perform numerous functions necessary for the survival of eukaryotic cells. These activities are coordinated by a diverse complement of proteins encoded in both the nuclear and mitochondrial genomes that must be properly organized and maintained. Misregulation of mitochondrial proteostasis impairs organellar function and can result in the development of severe human diseases. ATP-driven AAA+ proteins play crucial roles in preserving mitochondrial activity by removing and remodeling protein molecules in accordance with the needs of the cell. Two mitochondrial AAA proteases, i-AAA and m-AAA, are anchored to either face of the mitochondrial inner membrane, where they engage and process an array of substrates to impact protein biogenesis, quality control, and the regulation of key metabolic pathways. The functionality of these proteases is extended through multiple substrate-dependent modes of action, including complete degradation, partial processing, or dislocation from the membrane without proteolysis. This review discusses recent advances made toward elucidating the mechanisms of substrate recognition, handling, and degradation that allow these versatile proteases to control diverse activities in this multifunctional organelle.

  4. Conserved hydrogen bonds and water molecules in MDR HIV-1 protease substrate complexes

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Zhigang [Wayne State Univ., Detroit, MI (United States); Case Western Reserve Univ., Cleveland, OH (United States); Harbor Hospital Baltimore, MD (United States); Wang, Yong [Wayne State Univ., Detroit, MI (United States); Yedidi, Ravikiran S. [Wayne State Univ., Detroit, MI (United States); National Institutes of Health, Bethesda, MD (United States); Dewdney, Tamaria G. [Wayne State Univ., Detroit, MI (United States); Reiter, Samuel J. [Wayne State Univ., Detroit, MI (United States); Brunzelle, Joseph S. [Northwestern Univ. Feinberg School of Medicine, Chicago, IL (United States); Kovari, Iulia A. [Wayne State Univ., Detroit, MI (United States); Kovari, Ladislau C. [Wayne State Univ., Detroit, MI (United States)

    2012-12-19

    Success of highly active antiretroviral therapy (HAART) in anti-HIV therapy is severely compromised by the rapidly developing drug resistance. HIV-1 protease inhibitors, part of HAART, are losing their potency and efficacy in inhibiting the target. Multi-drug resistant (MDR) 769 HIV-1 protease (resistant mutations at residues 10, 36, 46, 54, 62, 63, 71, 82, 84, 90) was selected for the present study to understand the binding to its natural substrates. The nine crystal structures of MDR769 HIV-1 protease substrate hepta-peptide complexes were analyzed in order to reveal the conserved structural elements for the purpose of drug design against MDR HIV-1 protease. Our structural studies demonstrated that highly conserved hydrogen bonds between the protease and substrate peptides, together with the conserved crystallographic water molecules, played a crucial role in the substrate recognition, substrate stabilization and protease stabilization. Additionally, the absence of the key flap-ligand bridging water molecule might imply a different catalytic mechanism of MDR769 HIV-1 protease compared to that of wild type (WT) HIV-1 protease.

  5. Plant proteases for bioactive peptides release: A review.

    Science.gov (United States)

    Mazorra-Manzano, M A; Ramírez-Suarez, J C; Yada, R Y

    2017-04-10

    Proteins are a potential source of health-promoting biomolecules with medical, nutraceutical, and food applications. Nowadays, bioactive peptides production, its isolation, characterization, and strategies for its delivery to target sites are a matter of intensive research. In vitro and in vivo studies regarding the bioactivity of peptides has generated strong evidence of their health benefits. Dairy proteins are considered the richest source of bioactive peptides, however proteins from animal and vegetable origin also have been shown to be important sources. Enzymatic hydrolysis has been the process most commonly used for bioactive peptide production. Most commercial enzymatic preparations frequently used are from animal (e.g., trypsin and pepsin) and microbial (e.g., Alcalase® and Neutrase®) sources. Although the use of plant proteases is still relatively limited to papain and bromelain from papaya and pineapple, respectively, the application of new plant proteases is increasing. This review presents the latest knowledge in the use and diversity of plant proteases for bioactive peptides release from food proteins including both available commercial plant proteases as well as new potential plant sources. Furthermore, the properties of peptides released by plant proteases and health benefits associated in the control of disorders such as hypertension, diabetes, obesity, and cancer are reviewed.

  6. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

    Pesticide resistance is an example of evolution in action with mechanisms of resistance arising from mutations or increased expression of intrinsic genes. Intracellular proteases have a key role in maintaining healthy cells and in responding to stressors such as pesticides. Insecticide-resistant insects have constitutively elevated intracellular protease activity compared to corresponding susceptible strains. This increase was shown for some cases originally through biochemical enzyme studies and subsequently putatively by transcriptomics and proteomics methods. Upregulation and expression of proteases have been characterised in resistant strains of some insect species, including mosquitoes. This increase in proteolysis results in more degradation products (amino acids) of intracellular proteins. These may be utilised in the resistant strain to better protect the cell from stress. There are changes in insect intracellular proteases shortly after insecticide exposure, suggesting a role in stress response. The use of protease and proteasome inhibitors or peptide mimetics as synergists with improved application techniques and through protease gene knockdown using RNA interference (possibly expressed in crop plants) may be potential pest management strategies, in situations where elevated intracellular proteases are relevant. © 2017 Society of Chemical Industry. © 2017 Society of Chemical Industry.

  7. Co-evolution of insect proteases and plant protease inhibitors.

    Science.gov (United States)

    Jongsma, Maarten A; Beekwilder, Jules

    2011-08-01

    Plants are at the basis of the food chain, but there is no such thing as a "free lunch" for herbivores. To promote reproductive success, plants evolved multi-layered defensive tactics to avoid or discourage herbivory. To the detriment of plants, herbivores, in turn, evolved intricate strategies to find, eat, and successfully digest essential plant parts to raise their own offspring. In this battle the digestive tract is the arena determining final victory or defeat as measured by growth or starvation of the herbivore. Earlier, specific molecular opponents were identified as proteases and inhibitors: digestive proteases of herbivores evolved structural motifs to occlude plant protease inhibitors, or alternatively, the insects evolved proteases capable of specifically degrading the host plant inhibitors. In response plant inhibitors evolved hyper-variable and novel protein folds to remain active against potential herbivores. At the level of protease regulation in herbivorous insects, it was shown that inhibition-insensitive digestive proteases are up-regulated when sensitive proteases are inhibited. The way this regulation operates in mammals is known as negative feedback by gut-luminal factors, so-called 'monitor peptides' that are sensitive to the concentration of active enzymes. We propose that regulation of gut enzymes by endogenous luminal factors has been an open invitation to plants to "hijack" this regulation by evolving receptor antagonists, although yet these plant factors have not been identified. In future research the question of the co-evolution of insect proteases and plant inhibitors should, therefore, be better approached from a systems level keeping in mind that evolution is fundamentally opportunistic and that the plant's fitness is primarily improved by lowering the availability of essential amino acids to an herbivore by any available mechanism.

  8. Career Compromises: Framings and Their Implications.

    Science.gov (United States)

    Gati, Itamar; Houminer, Daphna; Aviram, Tamar

    1998-01-01

    Career compromise was investigated in three framings (alternatives, aspect importance, within-aspect preference). Young adults and school counselors rated hypothetical stories. Results of four studies with different designs (Average N=106) supported the hypothesis. The alternatives framing was associated with greater compromise and decision…

  9. Impending Airway Compromise due to Cystic Hygroma

    Directory of Open Access Journals (Sweden)

    Itai Shavit

    2011-05-01

    Full Text Available We report on a 3-month-old infant, who arrived in the pediatric emergency department (ED with a cervical cystic hygroma causing an impending compromise of the airway. We recognize that such a lesion can rapidly progress, and the judicious use of imaging in the ED may help to avoid airway compromise and possibly fatal complications. [West J Emerg Med. 2011;12(4:368–369.

  10. Influence of ensiling, exogenous protease addition, and bacterial inoculation on fermentation profile, nitrogen fractions, and ruminal in vitro starch digestibility in rehydrated and high-moisture corn.

    Science.gov (United States)

    Ferraretto, L F; Fredin, S M; Shaver, R D

    2015-10-01

    Exogenous protease addition may be an option to increase proteolysis of zein proteins and thus starch digestibility in rehydrated and high-moisture corn (HMC) ensiled for short periods. In addition, microbial inoculation may accelerate fermentation and increase acid production and thus increase solubilization of zein proteins. Four experiments were performed to evaluate the effect on fermentation profile, N fractions, and ruminal in vitro starch digestibility (ivSD) of the following: (1) rehydration and ensiling of dry ground corn; (2) exogenous protease addition to rehydrated un-ensiled and ensiled corn; (3) exogenous protease addition or inoculation in rehydrated ensiled corn; and (4) exogenous protease addition or inoculation in HMC. Experiments 1, 2, and 3 were performed with 7 treatments: dry ground corn (DGC); DGC rehydrated to a targeted dry matter content of 70% (REH); REH treated with exogenous protease (REH+); REH ensiled for 30 d (ENS); ENS treated with exogenous protease (ENS+); ENS treated with a microbial inoculant containing Lactobacillus plantarum, Lactobacillus casei, Enterococcus faecium, and Pediococcus sp. (ENSI); and ENS treated with exogenous protease and microbial inoculant (ENSI+). Experiment 1 compared DGC, REH, and ENS with ivSD being greater for ENS (64.9%) than DGC and REH (51.7% on average). Experiment 2 compared REH and ENS without or with exogenous protease addition (REH+ and ENS+, respectively). Ensiling and exogenous protease addition increased ivSD, but exogenous protease addition was more effective in ENS than REH (6.4 vs. 2.6 percentage unit increase). Experiment 3 compared the effects of exogenous protease addition and inoculation in ENS corn (ENS, ENS+, ENSI, and ENSI+). The addition of protease, but not inoculant, increased ivSD. Inoculation reduced pH and acetate, propionate, and ethanol concentrations, and increased lactate and total acid concentrations. In experiment 4, 8 treatments were a combination of HMC noninoculated

  11. Cytomegalovirus protease targeted prodrug development.

    Science.gov (United States)

    Sabit, Hairat; Dahan, Arik; Sun, Jing; Provoda, Chester J; Lee, Kyung-Dall; Hilfinger, John H; Amidon, Gordon L

    2013-04-01

    Human cytomegalovirus (HCMV) is a prevalent virus that infects up to 90% of the population. The goal of this research is to determine if small molecular prodrug substrates can be developed for a specific HCMV encoded protease and thus achieve site-specific activation. HCMV encodes a 256 amino acid serine protease that is responsible for capsid assembly, an essential process for herpes virus production. The esterase activity of the more stable HCMV A143T/A144T protease mutant was evaluated with model p-nitrophenol (ONp) esters, Boc-Xaa-ONp (Ala, Leu, Ile, Val, Gln, Phe at the Xaa position). We demonstrate that the A143T/A144T mutant has esterase activity toward specific small ester compounds, e.g., Boc-L-Ala-ONp. Mono amino acid and dipeptide prodrugs of ganciclovir (GCV) were also synthesized and evaluated for hydrolysis by the A143T/A144T protease mutant in solution. Hydrolysis of these prodrugs was also evaluated in Caco-2 cell homogenates, human liver microsomes (HLMs), and rat and human plasma. For the selectivity potential of the prodrugs, the hydrolysis ratio was evaluated as a percentage of prodrug hydrolyzed by the HCMV protease over the percentages of prodrug hydrolyses by Caco-2 cell homogenates, HLMs, and human/rat plasma. A dipeptide prodrug of ganciclovir, Ac-l-Gln-l-Ala-GCV, emerged as a potential selective prodrug candidate. The results of this research demonstrate that targeting prodrugs for activation by a specific protease encoded by the infectious HCMV pathogen may be achievable.

  12. Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria.

    Science.gov (United States)

    Sessenwein, Jessica L; Baker, Corey C; Pradhananga, Sabindra; Maitland, Megan E; Petrof, Elaine O; Allen-Vercoe, Emma; Noordhof, Curtis; Reed, David E; Vanner, Stephen J; Lomax, Alan E

    2017-11-29

    Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human

  13. Contemporary protease inhibitors and cardiovascular risk

    DEFF Research Database (Denmark)

    Lundgren, Jens; Mocroft, Amanda; Ryom, Lene

    2018-01-01

    PURPOSE OF REVIEW: To review the evidence linking use of HIV protease inhibitors with excess risk of cardiovascular disease (CVD) in HIV+ populations. RECENT FINDINGS: For the two contemporary most frequently used protease inhibitors, darunavir and atazanavir [both pharmacologically boosted...

  14. Dental management of medically compromised patients

    Directory of Open Access Journals (Sweden)

    Sherly Horax

    2016-06-01

    Full Text Available These days, treatment in dentistry is no longer for patient without complication, but also for patient with bad medical record. With correct treatment management in handling medical condition of patient, not only for dental treatment but also their systematic disease, all the dental staff also can improve for the better quality of life of the patient. Patient with medical compromised start to realize that  keeping good oral hygiene is so important for their lives, therefore dental staff need to improve their science and technology and also for facing patient with medical compromised. This article will discuss and suggest various treatment consideration and protocol for the patient of with medical compromised.

  15. A genomic survey of proteases in Aspergilli

    NARCIS (Netherlands)

    Budak, Sebnem Ozturkoglu; Zhou, M.; Brouwer, Carlo; Wiebenga, A.; Benoit, Isabelle; Di Falco, Marcos; Tsang, Adrian; de Vries, Ronald P; van den Brink, J.

    2014-01-01

    BACKGROUND: Proteases can hydrolyze peptides in aqueous environments. This property has made proteases the most important industrial enzymes by taking up about 60% of the total enzyme market. Microorganisms are the main sources for industrial protease production due to their high yield and a wide

  16. Curcumin derivatives as HIV-1 protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R. [Univ. of California, San Francisco, CA (United States)

    1993-12-31

    Curcumin, a non-toxic natural compound from Curcuma longa, has been found to be an HIV-1 protease inhibitor. Some of its derivatives were synthesized and their inhibitory activity against the HIV-1 protease was tested. Curcumin analogues containing boron enhanced the inhibitory activity. At least of the the synthesized compounds irreversibly inhibits the HIV-1 protease.

  17. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)

    oyaide

    2013-05-22

    May 22, 2013 ... suggest the suitability of the enzyme for applications in peptide synthesis, detergent formulation and ... The cell free supernatant was recovered as crude enzyme preparation and used for further studies. Assay of protease activity. Protease activity was ... Effect of pH on growth and protease production.

  18. Factor VII-activating protease

    DEFF Research Database (Denmark)

    Ramanathan, Ramshanker; Gram, Jørgen B; Sand, Niels Peter R

    2017-01-01

    : Factor VII-activating protease (FSAP) may regulate development of cardiovascular disease (CVD). We evaluated sex differences in FSAP measures and examined the association between FSAP and coronary artery calcification (CAC) in a middle-aged population. Participants were randomly selected citizens...

  19. Carbohydrase and protease supplementation increased ...

    African Journals Online (AJOL)

    A trial was conducted to evaluate whether the addition of commercial enzyme preparations containing carbohydrases and a protease would increase the available metabolizable energy (ME) of maize-soya-based broiler diets. Seven thousand five hundred and sixty (7560) day-old Ross 788 chicks were randomly allocated ...

  20. Evaluation of instant cup noodle, irradiated for immuno-compromised patients

    International Nuclear Information System (INIS)

    Lee, Ji-Hye; Kim, Jae-Kyung; Park, Jae-Nam; Yoon, Young-Min; Sung, Nak-Yun; Kim, Jae-Hun; Song, Beom-Seok; Yook, Hong-Sun; Kim, Byeong-Keun; Lee, Ju-Woon

    2012-01-01

    In the present study, initial microbial load of instant cup noodle (ICN) was investigated and gamma irradiation applied to develop immuno-compromised patients food for their safe consumption. The initial microbial population of dried vegetable and meat, and noodle was below the detection limit (1 log CFU/g); however, that of seasoning powder was just above 4 log CFU/g. Moreover, rehydrated-ICN with water at 100 °C still show above 3 log CFU/g of microbial load, which indicates the need for an additional process to control microbial safety of the seasoning powder. The total aerobic bacteria in seasoning powder and rehydrated-ICN could be controlled with 17 kGy gamma irradiation. This result referred 17 kGy gamma irradiation could reach ‘practical sterility’ of ICN. The overall difference in sensory properties between the non-irradiated and irradiated ICN was insignificant. Thus, gamma irradiation could improve the microbial quality of ICN, and reduce the risk of infection posed by the seasoning powder, without any adverse effects on their sensory quality. These results suggest that gamma-irradiated ICN can be used as a snack food for immuno-compromised patients.

  1. Evaluation of instant cup noodle, irradiated for immuno-compromised patients

    Science.gov (United States)

    Lee, Ji-Hye; Kim, Jae-Kyung; Park, Jae-Nam; Yoon, Young-Min; Sung, Nak-Yun; Kim, Jae-Hun; Song, Beom-Seok; Yook, Hong-Sun; Kim, Byeong-Keun; Lee, Ju-Woon

    2012-08-01

    In the present study, initial microbial load of instant cup noodle (ICN) was investigated and gamma irradiation applied to develop immuno-compromised patients food for their safe consumption. The initial microbial population of dried vegetable and meat, and noodle was below the detection limit (1 log CFU/g); however, that of seasoning powder was just above 4 log CFU/g. Moreover, rehydrated-ICN with water at 100 °C still show above 3 log CFU/g of microbial load, which indicates the need for an additional process to control microbial safety of the seasoning powder. The total aerobic bacteria in seasoning powder and rehydrated-ICN could be controlled with 17 kGy gamma irradiation. This result referred 17 kGy gamma irradiation could reach 'practical sterility' of ICN. The overall difference in sensory properties between the non-irradiated and irradiated ICN was insignificant. Thus, gamma irradiation could improve the microbial quality of ICN, and reduce the risk of infection posed by the seasoning powder, without any adverse effects on their sensory quality. These results suggest that gamma-irradiated ICN can be used as a snack food for immuno-compromised patients.

  2. Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases.

    Science.gov (United States)

    Lylloff, Jeanette E; Hansen, Lea B S; Jepsen, Morten; Sanggaard, Kristian W; Vester, Jan K; Enghild, Jan J; Sørensen, Søren J; Stougaard, Peter; Glaring, Mikkel A

    2016-03-01

    Proteases active at low temperature or high pH are used in many commercial applications, including the detergent, food and feed industries, and bacteria specifically adapted to these conditions are a potential source of novel proteases. Environments combining these two extremes are very rare, but offer the promise of proteases ideally suited to work at both high pH and low temperature. In this report, bacteria from two cold and alkaline environments, the ikaite columns in Greenland and alkaline ponds in the McMurdo Dry Valley region, Antarctica, were screened for extracellular protease activity. Two isolates, Arsukibacterium ikkense from Greenland and a related strain, Arsukibacterium sp. MJ3, from Antarctica, were further characterized with respect to protease production. Genome sequencing identified a range of potential extracellular proteases including a number of putative secreted subtilisins. An extensive liquid chromatography-tandem mass spectrometry analysis of proteins secreted by A. ikkense identified six subtilisin-like proteases as abundant components of the exoproteome in addition to other peptidases potentially involved in complete degradation of extracellular protein. Screening of Arsukibacterium genome libraries in Escherichia coli identified two orthologous secreted subtilisins active at pH 10 and 20 °C, which were also present in the A. ikkense exoproteome. Recombinant production of both proteases confirmed the observed activity. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  3. Strained Compromises? Danish Flexicurity During Crisis

    DEFF Research Database (Denmark)

    Ibsen, Christian Lyhne

    2011-01-01

    The Danish concept of flexicurity in a ‘Golden Triangle’ of low job protection, high income security and high employment security is not only about a balance between labor market flexibility and social security. Arguably, it is also a series of more or less stable underlying compromises between...

  4. Process optimization by response surface methodology for extracellular alkaline protease production from bacillus subtilis

    International Nuclear Information System (INIS)

    Mushtaq, Z.; Adnan, A.; Mehmood, Z.

    2014-01-01

    Three microbial cultures Bacillus subtilis DSM 1970, Bacillus subtilis GCU-8 and Bacillus licheniformis DSM 1969 were screened for protease production by casein agar plate method. Among these Bacillus subtilis GCU-8 was found to be the most potent protease producer in wide pH range (5.0 to 8.0). Fermentation conditions were optimized for the production of alkaline protease using two statistical tools: Placket Burmen Model for linear regression study and Response Surface Model for interactive effects of significant factors on production. The alkaline protease was optimally produced after 48 hours of incubation at 37 degree C in fermentation media containing equal amounts of substrates (soybean meal and wheat bran, 7.5 g), MgSO/sub 4/ 7H/sub 2/O, 0.10 g and yeast extract 0.55 g. The protease was purified to homogeneity by salt precipitation, ion-exchange chromatography and size exclusion chromatography. The homogeneity and molecular weights were checked by SDS-PAGE. The protease was 45 KDa protein, predominantly alkaline and optimally active at pH 8.0. (author)

  5. Protease-sensitive synthetic prions.

    Directory of Open Access Journals (Sweden)

    David W Colby

    2010-01-01

    Full Text Available Prions arise when the cellular prion protein (PrP(C undergoes a self-propagating conformational change; the resulting infectious conformer is designated PrP(Sc. Frequently, PrP(Sc is protease-resistant but protease-sensitive (s prions have been isolated in humans and other animals. We report here that protease-sensitive, synthetic prions were generated in vitro during polymerization of recombinant (rec PrP into amyloid fibers. In 22 independent experiments, recPrP amyloid preparations, but not recPrP monomers or oligomers, transmitted disease to transgenic mice (n = 164, denoted Tg9949 mice, that overexpress N-terminally truncated PrP. Tg9949 control mice (n = 174 did not spontaneously generate prions although they were prone to late-onset spontaneous neurological dysfunction. When synthetic prion isolates from infected Tg9949 mice were serially transmitted in the same line of mice, they exhibited sPrP(Sc and caused neurodegeneration. Interestingly, these protease-sensitive prions did not shorten the life span of Tg9949 mice despite causing extensive neurodegeneration. We inoculated three synthetic prion isolates into Tg4053 mice that overexpress full-length PrP; Tg4053 mice are not prone to developing spontaneous neurological dysfunction. The synthetic prion isolates caused disease in 600-750 days in Tg4053 mice, which exhibited sPrP(Sc. These novel synthetic prions demonstrate that conformational changes in wild-type PrP can produce mouse prions composed exclusively of sPrP(Sc.

  6. Objections to the Libertarian Stem Cell Compromise

    Directory of Open Access Journals (Sweden)

    Walter E. Block

    2010-11-01

    Full Text Available In Block (2010 I offered a compromise between the pro choice position that fervently supports stem cell research, and the pro life philosophy which bitterly opposes it. The compromise was a contest: allow would be researchers to create as many fertilized eggs as they wished. But, also, these should be offered up to would be parents to adopt all of these “children” as they wanted. If and only if there were any unadopted fetuses remaining in the laboratories of the nation would it be licit, on libertarian grounds, for research on them to take place. In the present paper I respond to several objections to this “modest proposal.”

  7. When data representation compromise data security

    DEFF Research Database (Denmark)

    Simonsen, Eivind Ortind; Dahl, Mads Ronald

    WHEN DATA REPRESENTATION COMPROMISE DATA SECURITY The workflow of transforming data into informative representations makes extensive usage of computers and software. Scientists have a conventional tradition for producing publications that include tables and graphs as data representations....... These representations can be used for multiple purposes such as publications in journals, teaching and conference material. But when created, stored and distributed in a digital form there is a risk of compromising data security. Data beyond the once used specifically to create the representation can be included...... on the internet over many years? A new legislation proposed in 2012 by the European Commission on protection of personal data will be implemented from 2015. The new law will impose sanction options ranging from a warning to a fine up to 100.000.000 EUR. We argue that this new law will lead to especially...

  8. Cysteine Protease Zymography: Brief Review.

    Science.gov (United States)

    Wilkesman, Jeff

    2017-01-01

    Cysteine proteases play multiple roles in basically all aspects of physiology and development. In plants, they are involved in growth and development and in accumulation and mobilization of storage proteins. Furthermore, they are engaged in signalling pathways and in the response to biotic and abiotic stresses. In animals and also in humans, they are responsible for senescence and apoptosis, prohormone processing, and ECM remodelling. When analyzed by zymography, the enzyme must be renaturated after SDS-PAGE. SDS must be washed out and substituted by Triton X-100. Gels are then further incubated under ideal conditions for activity detection. Cysteine proteases require an acidic pH (5.0-6.0) and a reducing agent, usually DTT. When screening biological samples, there is generally no previous clue on what peptidase class will be present, neither optimal proteolysis conditions are known. Hence, it is necessary to assess several parameters, such as incubation time, pH, temperature, influence of ions or reducing agents, and finally evaluate the inhibition profile. For detection of cysteine peptidase activity, the use of specific inhibitors, such as E-64, can be used to prevent the development of cysteine peptidase activity bands and positively confirm its presence. Here four different protocols to assess cysteine protease activity from different sources are presented.

  9. Production of Microbial Protease from Selected Soil Fungal Isolates

    African Journals Online (AJOL)

    Dr Oseni

    Aspergillus flavus, Aspergillus niger, Aspergillus fumigatus and Penicillium italicum produced the ... easier to be recovered from fermentation broth (Sandhya et al., 2005). .... and Penicillium italicum was observed for five days in liquid medium with 0.5% casein as a .... From the Thermophilic and Alkaliphilic Basillus spp.

  10. Advances in protease engineering for laundry detergents.

    Science.gov (United States)

    Vojcic, Ljubica; Pitzler, Christian; Körfer, Georgette; Jakob, Felix; Ronny Martinez; Maurer, Karl-Heinz; Schwaneberg, Ulrich

    2015-12-25

    Proteases are essential ingredients in modern laundry detergents. Over the past 30 years, subtilisin proteases employed in the laundry detergent industry have been engineered by directed evolution and rational design to tailor their properties towards industrial demands. This comprehensive review discusses recent success stories in subtilisin protease engineering. Advances in protease engineering for laundry detergents comprise simultaneous improvement of thermal resistance and activity at low temperatures, a rational strategy to modulate pH profiles, and a general hypothesis for how to increase promiscuous activity towards the production of peroxycarboxylic acids as mild bleaching agents. The three protease engineering campaigns presented provide in-depth analysis of protease properties and have identified principles that can be applied to improve or generate enzyme variants for industrial applications beyond laundry detergents. Copyright © 2015 Elsevier B.V. All rights reserved.

  11. Characterization of thermostable alkaline proteases from Bacillus infantis SKS1 isolated from garden soil.

    Directory of Open Access Journals (Sweden)

    Sandeep Kaur Saggu

    Full Text Available Proteases are one of the largest groups of hydrolytic enzymes constituting about 60% of total worldwide sales of industrial enzymes due to their wide applications in detergent, leather, textile, food and pharmaceutical industry. Microbial proteases have been preferred over animal and plant proteases because of their fundamental features and ease in production. Bacillus infantis SKS1, an alkaline protease producing bacteria has been isolated from garden soil of north India and identified using morphological, biochemical and molecular methods. 16S rDNA sequence amplified using universal primers has 99% sequence identity with corresponding gene sequence of Bacillus infantis strain FM 34 and Bacillus sp. Beige. The bacterial culture and its 16S rDNA gene sequence have been deposited to Microbial Culture Collection (Pune, India with accession number MCC 3035 and GenBank with accession number KR092197 respectively. The partially purified extract of Bacillus infantis SKS1 was thermostable and active in presence of Mg2+, acetyl acetone and laundry detergents implicating its application in industry. Production of these enzymes using this strain was maximized by optimization of various parameters including temperature, pH, media components and other growth conditions. Our results show that fructose and dextrose serve as the best carbon sources for production of these enzymes, highlighting the use of this strain for enzyme production utilizing relatively inexpensive substrates like beet molasses and corn steep liquor. Additionally, this strain showed maximum production of enzymes at 40°C similar to bacterial species used for commercial production of alkaline proteases. Characterization of alkaline proteases from this strain of Bacillus infantis and optimization of parameters for its production would help in understanding its industrial application and large-scale production.

  12. Extracellular proteases from Streptomyces phaeopurpureus ExPro138 inhibit spore adhesion, germination and appressorium formation in Colletotrichum coccodes.

    Science.gov (United States)

    Palaniyandi, S A; Yang, S H; Suh, J-W

    2013-07-01

    To study the antifungal mechanism of proteases from Streptomyces phaeopurpureus strain ExPro138 towards Colletotrichum coccodes and to evaluate its utilization as biofungicide. We screened proteolytic Streptomyces strains from the yam rhizosphere with antifungal activity. Forty proteolytic Streptomyces were isolated, among which eleven isolates showed gelatinolytic activity and antagonistic activity on C. coccodes. Of the 11 isolates, protease preparation from an isolate designated ExPro138 showed antifungal activity. 16S rDNA sequence analysis of the strain showed 99% similarity with Streptomyces phaeopurepureus (EU841588.1). Zymography analysis of the ExPro138 culture filtrate revealed that the strain produced several extracellular proteases. The protease preparation inhibited spore germination, spore adhesion to polystyrene surface and appressorium formation. Microscopic study of the interaction between ExPro138 and C. coccodes revealed that ExPro138 was mycoparasitic on C. coccodes. The protease preparation also reduced anthracnose incidence on tomato fruits compared with untreated control. This study demonstrates possibility of utilizing antifungal proteases derived from antagonistic microbes as biofungicide. Microbial proteases having the ability to inhibit spore adhesion and appressorium formation could be used to suppress infection establishment by foliar fungal pathogens at the initial stages of the infection process. Journal of Applied Microbiology © 2013 The Society for Applied Microbiology.

  13. 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.

  14. Strained Compromises? Danish Flexicurity During Crisis

    Directory of Open Access Journals (Sweden)

    Christian Lyhne Ibsen

    2011-01-01

    Full Text Available The Danish concept of flexicurity in a `Golden Triangle´ of low job protection, high income security and high employment security is not only about a balance between labor market flexibility and social security. Arguably, it is also a series of more or less stable underlying compromises between social partners about the main mechanisms and aims of labor market regulation which - supposedly - should be focused on employment rather than jobs, and competition on quality rather than on labor costs. However, the `Golden Triangle´ - this article argues - seems in need of complementary concepts. The article therefore introduces, `centralized decentralization´ - a concept that directs our attention to forms of flexibility and security primarily for people in work. Most studies on Danish flexicurity have been carried out under favorable economic conditions. In light of the economic slump hitting Denmark in 2008, this article investigates if and how the recession challenged these compromises by comparing two rounds of case-based interviews in three metalworking companies in 2007 and 2009. It is shown that practice has indeed changed - albeit modestly - due to worsened economic circumstances. For example the case studies show that the hypothesized preference for external numerical flexibility is perhaps too crude as employers use different ways to restructure employment. Especially the examples of de facto concessionary bargaining to save jobs are important here - although the extent of concessions is modest. The evidence thus suggests that the `Golden Triangle´ flexicurity compromises are indeed strained by the economic cycle and that responses to impetus for restructuring are far more nuanced than sometimes portrayed. It is argues that more company studies across national labor markets and industrial relations institutions will enhance our understanding of the dynamics during times of restructuring.

  15. How moral disagreement may ground principled moral compromise

    DEFF Research Database (Denmark)

    Kappel, Klemens

    2018-01-01

    In an influential article, Simon C. May forcefully argued that, properly understood, there can never be principled reasons for moral compromise (May, 2005). While there may be pragmatic reasons for compromising that involve, for instance, concern for political expediency or for stability, there a......In an influential article, Simon C. May forcefully argued that, properly understood, there can never be principled reasons for moral compromise (May, 2005). While there may be pragmatic reasons for compromising that involve, for instance, concern for political expediency or for stability......, there are properly speaking no principled reasons to compromise. My aim in the article is to show how principled moral compromise in the context of moral disagreements over policy options is possible. I argue that when we disagree, principled reasons favoring compromises or compromising can assume a more significant...... part of what makes a position all things considered best, and in this way disagreement can ground moral compromise....

  16. Optimization Conditions of Extracellular Proteases Production from a Newly Isolated Streptomyces Pseudogrisiolus NRC-15

    Directory of Open Access Journals (Sweden)

    El-Sayed E. Mostafa

    2012-01-01

    Full Text Available Microbial protease represents the most important industrial enzymes, which have an active role in biotechnological processes. The objective of this study was to isolate new strain of Streptomyces that produce proteolytic enzymes with novel properties and the development of the low-cost medium. An alkaline protease producer strain NRC-15 was isolated from Egyptian soil sample. The cultural, morphological, physiological characters and chemotaxonomic evidence strongly indicated that the NRC-15 strain represents a novel species of the genus Streptomyces, hence the name Strptomyces pseudogrisiolus NRC-15. The culture conditions for higher protease production by NRC-15 were optimized with respect to carbon and nitrogen sources, metal ions, pH and temperature. Maximum protease production was obtained in the medium supplemented with 1% glucose, 1% yeast extract, 6% NaCl and 100 μmol/L of Tween 20, initial pH 9.0 at 50 °C for 96 h. The current results confirm that for this strain, a great ability to produce alkaline proteases, which supports the use of applications in industry.

  17. Identifying genetic relatives without compromising privacy.

    Science.gov (United States)

    He, Dan; Furlotte, Nicholas A; Hormozdiari, Farhad; Joo, Jong Wha J; Wadia, Akshay; Ostrovsky, Rafail; Sahai, Amit; Eskin, Eleazar

    2014-04-01

    The development of high-throughput genomic technologies has impacted many areas of genetic research. While many applications of these technologies focus on the discovery of genes involved in disease from population samples, applications of genomic technologies to an individual's genome or personal genomics have recently gained much interest. One such application is the identification of relatives from genetic data. In this application, genetic information from a set of individuals is collected in a database, and each pair of individuals is compared in order to identify genetic relatives. An inherent issue that arises in the identification of relatives is privacy. In this article, we propose a method for identifying genetic relatives without compromising privacy by taking advantage of novel cryptographic techniques customized for secure and private comparison of genetic information. We demonstrate the utility of these techniques by allowing a pair of individuals to discover whether or not they are related without compromising their genetic information or revealing it to a third party. The idea is that individuals only share enough special-purpose cryptographically protected information with each other to identify whether or not they are relatives, but not enough to expose any information about their genomes. We show in HapMap and 1000 Genomes data that our method can recover first- and second-order genetic relationships and, through simulations, show that our method can identify relationships as distant as third cousins while preserving privacy.

  18. Optimization of alkaline protease production from Pseudomonas ...

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

    Dec 15, 2009 ... protease production was 37°C at pH 9, with 2% inoculum in the medium for 24 h. .... Positive. Catalase test. Positive ... The enzyme activity gradually decreases from ... Effect of temperature on protease production by Pseudomonas fluorescens. 0 .... between RNA polymerase and upstream promotes DNA.

  19. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    user

    2013-03-20

    Mar 20, 2013 ... Full Length Research Paper. Purification and ... ting into small peptides and free amino acids, which can ... Isolated strain was cultured in synthetic medium- casein (SMC; ... Protease activity was assayed by sigma's non-specific protease ... following buffers: 0.05 M citrate-phosphate buffer (pH 5 to 6), Tris-.

  20. Current and Novel Inhibitors of HIV Protease

    Czech Academy of Sciences Publication Activity Database

    Pokorná, Jana; Machala, L.; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    Roč. 1, č. 3 (2009), s. 1209-1239 ISSN 1999-4915 R&D Projects: GA MŠk 1M0508 Grant - others:GA AV ČR(CZ) IAAX00320901 Program:IA Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV protease * protease inhibitor * HAART Subject RIV: CE - Biochemistry

  1. No-compromise reptation quantum Monte Carlo

    International Nuclear Information System (INIS)

    Yuen, W K; Farrar, Thomas J; Rothstein, Stuart M

    2007-01-01

    Since its publication, the reptation quantum Monte Carlo algorithm of Baroni and Moroni (1999 Phys. Rev. Lett. 82 4745) has been applied to several important problems in physics, but its mathematical foundations are not well understood. We show that their algorithm is not of typical Metropolis-Hastings type, and we specify conditions required for the generated Markov chain to be stationary and to converge to the intended distribution. The time-step bias may add up, and in many applications it is only the middle of a reptile that is the most important. Therefore, we propose an alternative, 'no-compromise reptation quantum Monte Carlo' to stabilize the middle of the reptile. (fast track communication)

  2. Morgellons: contested illness, diagnostic compromise and medicalisation.

    Science.gov (United States)

    Fair, Brian

    2010-05-01

    The case of Morgellons illustrates how the emergence of a new medically contested illness intersected with and impacted on the diagnostic processes of an existing uncontested psychiatric condition, Delusional Parasitosis (DP). More specifically, the sociopolitical processes at play in the contested illness, Morgellons, dubiously reflect patient empowerment, as well the resilience and power of medical jurisdiction. This research offers insights into the contested illness and medicalisation literatures, and aims to bridge these two approaches towards the relationship between patient empowerment and medical authority, which I do through the notion of doctor-patient compromise. The data for this research come from a comprehensive qualitative analysis of Morgellons discourse through four key sources: the pro-Morgellons website Morgellons.org; the anti-Morgellons website Morgellonswatch.com; the popular media's portrayal of Morgellons; and the DP and Morgellons articles published in peer-reviewed medical journals, as made available on PubMed.

  3. Iran. Nuclear crisis: the continuous compromise?

    International Nuclear Information System (INIS)

    Cherief, Hamza

    2016-01-01

    The author comments and discusses the content and implications of the Joint Comprehensive Plan of Action which has been adopted in July 2015 by the Republic of Iran and the members of the E3/EU+3 group. According to this text, a flexible normative and institutional framework is defined, according to which negotiation must prevail on the reinstatement of economic sanctions. While significantly lowering objectives related to non proliferation, this plan aims (through various arrangements which are discussed by the author) at maintaining Iran under the threshold of nuclear power. The author also comments various aspects related to the implementation of this Plan of Action, and outlines that they rely on the search for a continuous compromise: negotiations and involvement of international bodies are foreseen before application of sanctions

  4. 6 CFR 13.46 - Compromise or settlement.

    Science.gov (United States)

    2010-01-01

    ... 6 Domestic Security 1 2010-01-01 2010-01-01 false Compromise or settlement. 13.46 Section 13.46 Domestic Security DEPARTMENT OF HOMELAND SECURITY, OFFICE OF THE SECRETARY PROGRAM FRAUD CIVIL REMEDIES § 13.46 Compromise or settlement. (a) Parties may Make offers of compromise or settlement at any time...

  5. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals.

    Science.gov (United States)

    Olajuyigbe, Folasade M; Demitri, Nicola; De Zorzi, Rita; Geremia, Silvano

    2016-10-31

    Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  6. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals

    Directory of Open Access Journals (Sweden)

    Folasade M. Olajuyigbe

    2016-10-01

    Full Text Available Protease inhibitors are key components in the chemotherapy of HIV infection. However, the appearance of viral mutants routinely compromises their clinical efficacy, creating a constant need for new and more potent inhibitors. Recently, a new class of epoxide-based inhibitors of HIV-1 protease was investigated and the configuration of the epoxide carbons was demonstrated to play a crucial role in determining the binding affinity. Here we report the comparison between three crystal structures at near-atomic resolution of HIV-1 protease in complex with the epoxide-based inhibitor, revealing an in-situ epoxide ring opening triggered by a pH change in the mother solution of the crystal. Increased pH in the crystal allows a stereospecific nucleophile attack of an ammonia molecule onto an epoxide carbon, with formation of a new inhibitor containing amino-alcohol functions. The described experiments open a pathway for the development of new stereospecific protease inhibitors from a reactive lead compound.

  7. Sterile diets for the immuno-compromised: Is there a need?

    Science.gov (United States)

    Butterweck, Joseph S.

    1995-02-01

    There is a general misunderstanding in the radiation processing industry about the use of sterile diets in the medical profession. Sterile diets are used on a limited basis in hospitals that specialize in cancer treatment and organ transplants. These patients are severely immuno-compromised. There are many other patients that are immuno-compromised that do not require sterile diets. These patients may require a diet that is pathogen-free and are aslo "low-microbial diets". Nosocomial infections have become a major issue in US hospitals. The "infection control committee" is the focus group responsible to assure nosocomial infections incidence are below the hospital goals. Application of ionizing radiation to sterilize diets has not been chosen because the product is not available at a reasonable total cost. This paper will discuss the hospitals views.

  8. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

    Magdolen, U.; Krol, J.; Sato, S.; Schmitt, M.; Magdolen, V.; Krueger, A.; Mueller, M.M.; Sperl, S.

    2002-01-01

    The turnover and remodelling of extracellular matrix (ECM) is an essential part of many normal biological processes including development, morphogenesis, and wound healing. ECM turnover also occurs in severe pathological situations like artherosclerosis, fibrosis, tumor invasion and metastasis. The major proteases involved in this turnover are serine proteases (especially the urokinase-type plasminogen activator/plasmin system), matrix metalloproteases (a family of about 20 zinc-dependent endopeptidases including collagenases, gelatinases, stromelysins, and membrane-type metalloproteases), and cysteine proteases. In vivo, the activity of these proteases is tightly regulated in the extracellular space by zymogen activation and/or controlled inhibition. In the present review, we give an overview on the structure and biochemical properties of important tumor-associated protease inhibitors such as plasminogen activator inhibitor type 1 and type 2 (PAI-1, PAI-2), tissue inhibitors of metalloproteinases (TIMP-1, -2, -3, and -4), and the cysteine protease inhibitor cystatin C. Interestingly, some of these inhibitors of tumor-associated proteases display multiple functions which rather promote than inhibit tumor progression, when the presence of inhibitors in the tumor tissue is not balanced. (author)

  9. Extracellular proteases of Trichoderma species. A review.

    Science.gov (United States)

    Kredics, L; Antal, Zsuzsanna; Szekeres, A; Hatvani, L; Manczinger, L; Vágvölgyi, Cs; Nagy, Erzsébet

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed.

  10. Gut proteases target Yersinia invasin in vivo

    Directory of Open Access Journals (Sweden)

    Freund Sandra

    2011-04-01

    Full Text Available Abstract Background Yersinia enterocolitica is a common cause of food borne gastrointestinal disease. After oral uptake, yersiniae invade Peyer's patches of the distal ileum. This is accomplished by the binding of the Yersinia invasin to β1 integrins on the apical surface of M cells which overlie follicle associated lymphoid tissue. The gut represents a barrier that severely limits yersiniae from reaching deeper tissues such as Peyer's patches. We wondered if gut protease attack on invasion factors could contribute to the low number of yersiniae invading Peyer's patches. Findings Here we show that invasin is rapidly degraded in vivo by gut proteases in the mouse infection model. In vivo proteolytic degradation is due to proteolysis by several gut proteases such as trypsin, α-chymotrypsin, pancreatic elastase, and pepsin. Protease treated yersiniae are shown to be less invasive in a cell culture model. YadA, another surface adhesin is cleaved by similar concentrations of gut proteases but Myf was not cleaved, showing that not all surface proteins are equally susceptible to degradation by gut proteases. Conclusions We demonstrate that gut proteases target important Yersinia virulence factors such as invasin and YadA in vivo. Since invasin is completely degraded within 2-3 h after reaching the small intestine of mice, it is no longer available to mediate invasion of Peyer's patches.

  11. Conflicting perspectives compromising discussions on cardiopulmonary resuscitation.

    LENUS (Irish Health Repository)

    Groarke, J

    2010-09-01

    Healthcare professionals, patients and their relatives are expected to discuss resuscitation together. This study aims to identify the differences in the knowledge base and understanding of these parties. Questionnaires examining knowledge and opinion on resuscitation matters were completed during interviews of randomly selected doctors, nurses and the general public. 70% doctors, 24% nurses and 0% of a public group correctly estimated survival to discharge following in-hospital resuscitation attempts. Deficiencies were identified in doctor and nurse knowledge of ethics governing resuscitation decisions. Public opinion often conflicts with ethical guidelines. Public understanding of the nature of cardiopulmonary arrests and resuscitation attempts; and of the implications of a \\'Do Not Attempt Resuscitation (DNAR)\\' order is poor. Television medical dramas are the primary source of resuscitation knowledge. Deficiencies in healthcare professionals\\' knowledge of resuscitation ethics and outcomes may compromise resuscitation decisions. Educational initiatives to address deficiencies are necessary. Parties involved in discussion on resuscitation do not share the same knowledge base reducing the likelihood of meaningful discussion. Public misapprehensions surrounding resuscitation must be identified and corrected during discussion.

  12. Orthodontic management of a periodontally compromised dentition

    Directory of Open Access Journals (Sweden)

    Prashant K Zaveri

    2016-01-01

    Full Text Available Malocclusion superimposed with severe periodontitis may present a great challenge to clinicians while providing orthodontic treatment due the episodic and site-specific nature of the disease with risk of rapid tissue breakdown. However, orthodontic treatment in such situation may contribute significantly to the overall rehabilitation both functionally and esthetically. In this article, a case report outlines a combined periodontic-orthodontic management of compromised dentition. A 37-year-old female patient with significant medical history was treated for Class II Division 1 type of malocclusion associated with spaced upper and lower anterior teeth, deep overbite, and increased overjet, superimposed with chronic generalized periodontitis and bone loss. Treatment was completed using temporary anchorage devices assisted strategically applied force and modified tandem retraction biomechanics amidst management of acute inflammatory episodes during and mucogingival complication after treatment. Affected areas healed very well after post-orthodontic periodontal treatment with minimal pocket depth, and bleeding on probing, and a healthy zone of attached gingiva at the follow up visits. The orthodontic results lead to improvement in patient's facial profile, lip posture, and correction of protrusion which addressed her main concern. One year follow-up shows good orthodontic and periodontic stability. The report highlights the importance of identifying “at risk” individuals and continuous monitoring of disease status during treatment. Despite all precautionary measures, a flare-up during the treatment can be anticipated.

  13. Protease and protease inhibitory activity in pregnant and postpartum involuting uterus

    International Nuclear Information System (INIS)

    Milwidsky, A.; Beller, U.; Palti, Z.; Mayer, M.

    1982-01-01

    The presence of two distinct proteolytic activities in the rat uterus was confirmed with 14 C-labeled globin used as a sensitive protein substrate and following release of label into the trichloroacetic acid-soluble supernatant fraction. Protease I is a cytoplasmic acid protease while protease II is associated with the pellet fraction, can be extracted by 0.6 M sodium chloride, and is active at pH 7.0. Protease I activity is low during pregnancy and markedly increases at term achieving maximal activity at day 3 post partum with a subsequent decline to preterm activity values. Lactation did not affect the uterine protease I activity. Protease II activity is not significantly different during pregnancy, at term, and post partum. The presence of an inhibitor of protease I was suggested by a decrease in enzyme activity with an increased cytosolic protein concentration. The inhibitor also lessened bovine trypsin activity but had no effect on protease II. Although its inhibitory potency on trypsin fluctuated during the various uterine physiologic stages, these changes appeared to be statistically insignificant. Human uterine samples were also found to contain the two protease activities with similar changes in protease I post partum. It is suggested that, both in the rat and in man, uterine involution post partum is associated with a marked increase in activity of acid cytosolic protease, while a particulate neutral protease and a soluble inhibitor of trypsin, which are also present in uterine cells, do not appear to play a significant role in the dissolution of uterine tissues after parturition

  14. Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography.

    Science.gov (United States)

    Kernaghan, Gavin; Mayerhofer, Michael

    2017-01-01

    This chapter describes a method for the production and characterization of fungal acid proteases. Protease production is induced by growth on BSA media over a pH gradient and protein levels are monitored over time with the Bradford assay. Once protein is depleted, the media is purified and proteases are characterized by gelatin zymography using acrylamide and buffers at near-neutral pH. Maintaining pH levels below those found in traditional zymographic systems avoids the potential loss of activity that may occur in aspartic proteases under alkaline conditions.

  15. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.

    2001-01-01

    Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency: elimina......Conversion of latent proteases to the active form occurs by various mechanisms characteristic for different protease families. Here we report that the disintegrin metalloprotease ADAM 12-S is activated by Cu(II). Copper activation is distinct from the cysteine switch component of latency......: elimination of the ADAM 12 cysteine switch by a point mutation in the propeptide had no effect on copper activation, whereas mutation of an unpaired cysteine residue in the catalytic domain resulted in a mutant form of ADAM 12-S that was insensitive to copper. This suggests a multi-step activation mechanism...... for ADAM 12 involving both furin cleavage and copper binding....

  16. Optimization of medium composition for thermostable protease ...

    African Journals Online (AJOL)

    SERVER

    2008-04-17

    Apr 17, 2008 ... Optimization of the fermentation medium for maximization of thermostable neutral protease production by Bacillus sp. ..... Each contour curve represented an infinite number of combinations of two ..... Production in sea-water of.

  17. Partial Purification and Characterization of Extracellular Protease ...

    African Journals Online (AJOL)

    USER

    Keywords: Protease, lactic acid bacteria, Pediococcus acidilactici, enzyme ... confers organoleptic improvements in fermented foods ... was characterized by studying the effect of substrate ... addition of solid ammonium sulphate up to 80%.

  18. Purification and characterization of protease enzyme from ...

    African Journals Online (AJOL)

    The enzyme was active in pH range 5 to11 and temperature of 30 to 80°C. The optimum pH and the temperature for protease activity were recorded to be pH 8 and 50°C, respectively. The enzyme was stable up to 40°C and pH 9. The protease activity was inhibited by Zn2+, Ni2+ and Sn2+ and increased by Ca2+, Mg2+ ...

  19. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. [Ohio State Univ., Columbus, OH (United States)]|[Lawrence Berkeley Lab., CA (United States)

    1995-12-31

    The synthesis of oligopeptides using stable carbohydrate protease conjugates (CPCs) was examined in acetonitrile solvent systems. CPC[{alpha}-chymotrypsin] was used for the preparation of peptides containing histidine, phenylalanine, tryptophan in the P{sub 1} position in 60-93% yield. The CPC[{alpha}-chymotrypsin]-catalyzed synthesis of octamer Z-Gly-Gly-Phe-Gly-Gly-Phe-Gly-Gly-OEt from Z-Gly-Gly-Phe-Gly-Gly-Phe-OMe was achieved in 71% yield demonstrating that synthesis peptides containing both hydrophylic and hydrophobic amino acids. The P{sub 2} specificity of papain for aromatic residues was utilized for the 2 + 3 coupling of Z-Tyr-Gly-OMe to H{sub 2}N-Gly-Phe-Leu-OH to generate the leucine enkephalin derivative in 79% yield. Although papain is nonspecific for the hydrolysis of N-benzyloxycarbonyl amino acid methyl esters in aqueous solution, the rates of synthesis for these derivitives with nucleophile leucine tert-butyl ester differed by nearly 2 orders of magnitude. CPC[thermolysin] was used to prepare the aspartame precursor Z-Asp-Phe-OMe in 90% yield. The increased stability of CPCs prepared from periodate-modified poly(2-methacryl- amido-2-deoxy-D-glucose), poly(2-methacrylamido-2-deoxy-D-galactose), and poly(5-methacryl-amido-5-deoxy-D-ribose), carbohydrate materials designed to increase the aldehyde concentration in aqueous solution, suggests that the stability of CPCs is directly related to the aldehyde concentration of the carbohydrate material. Periodate oxidation of poly(2-methacrylamido-2-deoxy-D-glucose) followed by covalent attachment to {alpha}-chymotrypsin gave a CPC with catalytic activity in potassium phosphate buffer at 90{degrees}C for 2 h. 1 fig., 1 tab., 40 refs.

  20. Compromise in cooperative game and the VIKOR method

    Directory of Open Access Journals (Sweden)

    Opricović Serafim

    2009-01-01

    Full Text Available Five approaches in conflict resolution are distinguished, based on cooperativeness and aggressiveness in resolving conflict. Compromise based on cooperativeness is emphasized here as a solution in conflict resolution. Cooperative game theory oriented towards aiding the conflict resolution is considered and the compromise value for TU(transferable utility-game is presented. The method VIKOR could be applied to determine compromise solution of a multicriteria decision making problem with noncommensurable and conflicting criteria. Compromise is considered as an intermediate state between conflicting objectives or criteria reached by mutual concession. The applicability of the cooperative game theory and the VIKOR method for conflict resolution is illustrated.

  1. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    Energy Technology Data Exchange (ETDEWEB)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-08-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon/sup -/ cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes (/sup 3/H)methyl-casein to acid-soluble products in the presence of ATP and Mg/sup 2 +/. ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles.

  2. Escherichia coli contains a soluble ATP-dependent protease (Ti) distinct from protease La

    International Nuclear Information System (INIS)

    Hwang, B.J.; Park, W.J.; Chung, C.H.; Goldberg, A.L.

    1987-01-01

    The energy requirement for protein breakdown in Escherichia coli has generally been attributed to the ATP-dependence of protease La, the lon gene product. The authors have partially purified another ATP-dependent protease from lon - cells that lack protease La (as shown by immunoblotting). This enzyme hydrolyzes [ 3 H]methyl-casein to acid-soluble products in the presence of ATP and Mg 2+ . ATP hydrolysis appears necessary for proteolytic activity. Since this enzyme is inhibited by diisopropyl fluorophosphate, it appears to be a serine protease, but it also contains essential thiol residues. They propose to name this enzyme protease Ti. It differs from protease La in nucleotide specificity, inhibitor sensitivity, and subunit composition. On gel filtration, protease Ti has an apparent molecular weight of 370,000. It can be fractionated by phosphocellulose chromatography or by DEAE chromatography into two components with apparent molecular weights of 260,000 and 140,000. When separated, they do not show preteolytic activity. One of these components, by itself, has ATPase activity and is labile in the absence of ATP. The other contains the diisopropyl fluorophosphate-sensitive proteolytic site. These results and the similar findings of Katayama-Fujimura et al. indicate that E. coli contains two ATP-hydrolyzing proteases, which differ in many biochemical features and probably in their physiological roles

  3. Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation

    DEFF Research Database (Denmark)

    Kousted, Tina Mostrup; Skjoedt, K; Petersen, S V

    2013-01-01

    abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between...

  4. Purification and characterisation of a protease (tamarillin) from tamarillo fruit

    KAUST Repository

    Li, Zhao

    2018-02-16

    A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named \\'tamarillin\\'.

  5. Purification and characterisation of a protease (tamarillin) from tamarillo fruit

    KAUST Repository

    Li, Zhao; Scott, Ken; Hemar, Yacine; Zhang, Huoming; Otter, Don

    2018-01-01

    A protease from tamarillo fruit (Cyphomandra betacea Cav.) was purified by ammonium sulphate precipitation and diethylaminoethyl-Sepharose chromatography. Protease activity was determined on selected peak fractions using a casein substrate. Sodium dodecyl sulphate polyacrylamide gel electrophoresis analysis showed that the peak with the highest protease activity consisted of one protein of molecular mass ca. 70 kDa. The protease showed optimal activity at pH 11 and 60°C. It was sensitive to phenylmethylsulphonyl fluoride while ethylenediaminetetraacetic acid and p-chloromercuribenzoic acid had little effect on its activity, indicating that this enzyme was a serine protease. Hg2+ strongly inhibited enzyme activity, possibly due to formation of mercaptide bonds with the thiol groups of the protease, suggesting that some cysteine residues may be located close to the active site. De novo sequencing strongly indicated that the protease was a subtilisin-like alkaline serine protease. The protease from tamarillo has been named 'tamarillin'.

  6. 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.

  7. [Protease activity of microflora in the oral cavity of patients with periodontitis].

    Science.gov (United States)

    Voropaeva, E A; Baĭrakova, A L; Bichucher, A M; D'iakov, V L; Kozlov, L V

    2008-01-01

    Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.

  8. 41 CFR 105-55.019 - Compromise of claims.

    Science.gov (United States)

    2010-07-01

    ... and a recommendation for the acceptance of the compromise offer. Justice Department approval is not... exercise the authorities in this section. (b) Unless otherwise provided by law, when the principal balance... Department of Justice. GSA will evaluate the compromise offer, using the factors set forth in § 105-55.020...

  9. 22 CFR 213.25 - Standards for compromise.

    Science.gov (United States)

    2010-04-01

    ... proceedings. In evaluating the acceptability of the offer, the CFO may consider, among other factors, the... applicable exemptions available to the debtor under State and Federal law in determining the Government's ability to enforce collection. (b) USAID may compromise a claim, or recommend acceptance of a compromise...

  10. 20 CFR 255.18 - Compromise of overpayments.

    Science.gov (United States)

    2010-04-01

    ... 20 Employees' Benefits 1 2010-04-01 2010-04-01 false Compromise of overpayments. 255.18 Section 255.18 Employees' Benefits RAILROAD RETIREMENT BOARD REGULATIONS UNDER THE RAILROAD RETIREMENT ACT... standards which the Board applies in exercising its authority under 31 U.S.C. 3711 to compromise an...

  11. 32 CFR 310.50 - Lost, stolen, or compromised information.

    Science.gov (United States)

    2010-07-01

    ... Official for Privacy within 24 hours of discovering that a breach of personally identifiable information... Privacy Office of the breach within 48 hours upon being notified that a loss, theft, or compromise has... (CONTINUED) PRIVACY PROGRAM DOD PRIVACY PROGRAM Privacy Act Violations § 310.50 Lost, stolen, or compromised...

  12. 16 CFR 1.96 - Compromise of penalty.

    Science.gov (United States)

    2010-01-01

    ... 16 Commercial Practices 1 2010-01-01 2010-01-01 false Compromise of penalty. 1.96 Section 1.96 Commercial Practices FEDERAL TRADE COMMISSION ORGANIZATION, PROCEDURES AND RULES OF PRACTICE GENERAL... may compromise any penalty or proposed penalty at any time, with leave of court when necessary, taking...

  13. Establishment of normal gut microbiota is compromised under excessive hygiene conditions.

    Directory of Open Access Journals (Sweden)

    Bettina Schmidt

    Full Text Available BACKGROUND: Early gut colonization events are purported to have a major impact on the incidence of infectious, inflammatory and autoimmune diseases in later life. Hence, factors which influence this process may have important implications for both human and animal health. Previously, we demonstrated strong influences of early-life environment on gut microbiota composition in adult pigs. Here, we sought to further investigate the impact of limiting microbial exposure during early life on the development of the pig gut microbiota. METHODOLOGY/PRINCIPAL FINDINGS: Outdoor- and indoor-reared animals, exposed to the microbiota in their natural rearing environment for the first two days of life, were transferred to an isolator facility and adult gut microbial diversity was analyzed by 16S rRNA gene sequencing. From a total of 2,196 high-quality 16S rRNA gene sequences, 440 phylotypes were identified in the outdoor group and 431 phylotypes in the indoor group. The majority of clones were assigned to the four phyla Firmicutes (67.5% of all sequences, Proteobacteria (17.7%, Bacteroidetes (13.5% and to a lesser extent, Actinobacteria (0.1%. Although the initial maternal and environmental microbial inoculum of isolator-reared animals was identical to that of their naturally-reared littermates, the microbial succession and stabilization events reported previously in naturally-reared outdoor animals did not occur. In contrast, the gut microbiota of isolator-reared animals remained highly diverse containing a large number of distinct phylotypes. CONCLUSIONS/SIGNIFICANCE: The results documented here indicate that establishment and development of the normal gut microbiota requires continuous microbial exposure during the early stages of life and this process is compromised under conditions of excessive hygiene.

  14. Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

    Directory of Open Access Journals (Sweden)

    Rachana Fulzele

    2011-12-01

    Full Text Available Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i motile ii rod shaped iii non spore forming iv catalase and amylase positive v able to grow in presence of 10 % NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7 exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98% sequence similarity, 1201 bp. The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 % activity at 80 0C after 4 h and more than 70 % activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 % active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

  15. Serine protease inhibitors of parasitic helminths.

    Science.gov (United States)

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.

  16. Tunable protease-activatable virus nanonodes.

    Science.gov (United States)

    Judd, Justin; Ho, Michelle L; Tiwari, Abhinav; Gomez, Eric J; Dempsey, Christopher; Van Vliet, Kim; Igoshin, Oleg A; Silberg, Jonathan J; Agbandje-McKenna, Mavis; Suh, Junghae

    2014-05-27

    We explored the unique signal integration properties of the self-assembling 60-mer protein capsid of adeno-associated virus (AAV), a clinically proven human gene therapy vector, by engineering proteolytic regulation of virus-receptor interactions such that processing of the capsid by proteases is required for infection. We find the transfer function of our engineered protease-activatable viruses (PAVs), relating the degree of proteolysis (input) to PAV activity (output), is highly nonlinear, likely due to increased polyvalency. By exploiting this dynamic polyvalency, in combination with the self-assembly properties of the virus capsid, we show that mosaic PAVs can be constructed that operate under a digital AND gate regime, where two different protease inputs are required for virus activation. These results show viruses can be engineered as signal-integrating nanoscale nodes whose functional properties are regulated by multiple proteolytic signals with easily tunable and predictable response surfaces, a promising development toward advanced control of gene delivery.

  17. Microbial biosensors

    International Nuclear Information System (INIS)

    Le Yu; Chen, Wilfred; Mulchandani, Ashok

    2006-01-01

    A microbial biosensor is an analytical device that couples microorganisms with a transducer to enable rapid, accurate and sensitive detection of target analytes in fields as diverse as medicine, environmental monitoring, defense, food processing and safety. The earlier microbial biosensors used the respiratory and metabolic functions of the microorganisms to detect a substance that is either a substrate or an inhibitor of these processes. Recently, genetically engineered microorganisms based on fusing of the lux, gfp or lacZ gene reporters to an inducible gene promoter have been widely applied to assay toxicity and bioavailability. This paper reviews the recent trends in the development and application of microbial biosensors. Current advances and prospective future direction in developing microbial biosensor have also been discussed

  18. Production of alkaline proteases by alkalophilic Bacillus subtilis ...

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

    Nov 23, 2016 ... Key words: Production, alkaline protease, Bacillus subtilis, animal wastes, enzyme activity. ... Generally, alkaline proteases are produced using submerged fermentation .... biopolymer concentrations were reported to have an influence ... adding nitrogenous compounds stimulate microorganism growth and ...

  19. Optimization of alkaline protease production and its fibrinolytic ...

    African Journals Online (AJOL)

    Optimization of alkaline protease production and its fibrinolytic activity from the ... nitrogen sources and sodium chloride concentration for protease production by the ... exploited to assist in protein degradation in various industrial processes.

  20. Purification and characterization of protease from Bacillus cereus ...

    African Journals Online (AJOL)

    Among them, SU12 isolate was selected due to its high enzyme production ... growth and protease production which includes different carbon and nitrogen sources, ... organism for the industrial production of the extracellular protease enzyme.

  1. Mosaic serine proteases in the mammalian central nervous system.

    Science.gov (United States)

    Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

    2008-01-01

    We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

  2. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    DEFF Research Database (Denmark)

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

    En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner.......En litteraturgennemgang af muligheder for at bruge cystein proteaser som antigener i antiparasitære vacciner....

  3. Compromised data from social media to big data

    CERN Document Server

    Redden, Joanna; Langlois, Ganaele

    2015-01-01

    There has been a data rush in the past decade brought about by online communication and, in particular, social media (Facebook, Twitter, Youtube, among others), which promises a new age of digital enlightenment. But social data is compromised: it is being seized by specific economic interests, it leads to a fundamental shift in the relationship between research and the public good, and it fosters new forms of control and surveillance. Compromised Data: From Social Media to Big Data explores how we perform critical research within a compromised social data framework. The expert, international l

  4. Pengaruh PH dan Suhu terhadap Aktivitas Protease Penicillium SP.

    OpenAIRE

    Yusriah, Yusriah; Kuswytasari, Nengah Dwianita

    2013-01-01

    Tujuan penelitian ini adalah untuk mengetahui pengaruh pH dan suhu terhadap aktivitas protease pada Penicillium sp.3 T3f2. Selanjutnya, isolat Penicillium sp. di kultur dalam media produksi protease untuk menghasilkan protease. Suhu yang digunakan adalah 300 – 500C sedangkan pH-nya 4 – 8. Aktivitas protease ditentukan dan diukur dengan spektrofotometer pada panjang gelombang 275 nm, dengan kasein sebagai substrat. Berdasarkan uji ANOVA yang dilanjutkan dengan uji Duncan dengan taraf kepercaya...

  5. Protease inhibitor (PI) mediated defense in leaves and flowers of pigeonpea (protease inhibitor mediated defense in pigeonpea).

    Science.gov (United States)

    Padul, Manohar V; Tak, Rajesh D; Kachole, Manvendra S

    2012-03-01

    More than 200 insect pests are found growing on pigeonpea. Insects lay eggs, attack and feed on leaves, flowers and developing pods. Plants have developed elaborate defenses against these insect pests. The present work evaluates protease inhibitor (PI) based defense of pigeonpea in leaves and flowers. PIs in the extracts of these tender tissues were detected by using gel X-ray film contact print method. Up to three PIs (PI-3, PI-4 and PI-5) were detected in these tissues as against nine (PI-1-PI-9) in mature seeds. PI-3 is the major component of these tissues. Mechanical wounding, insect chewing, fungal pathogenesis and application of salicylic acid induced PIs in pigeonpea in these tissues. Induction was found to be local as well as systemic but local response was stronger than systemic response. During both local and systemic induction, PI-3 appeared first. In spite of the presence and induction of PIs in these tender tissues and seeds farmers continue to suffer yield loses. This is due to the weak expression of PIs. However the ability of the plant to respond to external stimuli by producing defense proteins does not seem to be compromised. This study therefore indicates that PIs are components of both constitutive and inducible defense and provide a ground for designing stronger inducible defense (PIs or other insect toxin based) in pigeonpea. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  6. 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

  7. Lipase and protease extraction from activated sludge

    DEFF Research Database (Denmark)

    Gessesse, Amare; Dueholm, Thomas; Petersen, Steffen B.

    2003-01-01

    of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination...

  8. HIV-1 protease-induced apoptosis

    Czech Academy of Sciences Publication Activity Database

    Rumlová, Michaela; Křížová, Ivana; Keprová, Alena; Hadravová, Romana; Doležal, Michal; Strohalmová, Karolína; Pichová, Iva; Hájek, Miroslav; Ruml, T.

    2014-01-01

    Roč. 11, May 20 (2014), 37/1-37/15 ISSN 1742-4690 R&D Projects: GA ČR GA204/09/1388 Institutional support: RVO:61388963 Keywords : HIV protease * BCA3 * AKIP-1 * apoptosis * mitochondria Subject RIV: EE - Microbiology, Virology Impact factor: 4.185, year: 2014 http://www.retrovirology.com/content/11/1/37

  9. Bacterial proteases: targets for diagnostics and therapy

    NARCIS (Netherlands)

    Kaman, W.E.; Hays, J.P.; Endtz, H.P.; Bikker, F.J.

    2014-01-01

    Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and

  10. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    of novel peptide-based protease inhibitors, efforts were made towards improved methods for peptide synthesis. The coupling of Fmoc-amino acids onto N-methylated peptidyl resins was investigated. These couplings can be low yielding and the effect of the use of microwave heating combined with the coupling...

  11. The role of postoperative hematoma on free flap compromise.

    Science.gov (United States)

    Ahmad, Faisal I; Gerecci, Deniz; Gonzalez, Javier D; Peck, Jessica J; Wax, Mark K

    2015-08-01

    Hematomas may develop in the postoperative setting after free tissue transfer. When hematomas occur, they can exert pressure on surrounding tissues. Their effect on the vascular pedicle of a free flap is unknown. We describe our incidence of hematoma in free flaps and outcomes when the flap is compromised. Retrospective chart review of 1,883 free flaps performed between July 1998 and June 2014 at a tertiary referral center. Patients with free flap compromise due to hematoma were identified. Etiology, demographic data, and outcomes were evaluated. Eighty-eight (4.7%) patients developed hematomas. Twenty (22.7%) of those had flap compromise. Twelve compromises (60%) showed evidence of pedicle thrombosis. The salvage rate was 75% versus 54% in 79 flaps with compromise from other causes (P = .12). Mean time to detection of the hematoma was 35.3 hours in salvaged flaps compared to 91.6 hours in unsalvageable flaps (P = .057). Time to operating room (OR) from detection was 2.8 hours in salvageable flaps compared to 12.4 hours in nonsalvageable flaps (P = .053). The salvage rate for flaps that returned to the OR in hematomas developed rarely. When they did, 23% went on to develop flap compromise. Prompt recognition and re-exploration allowed for a high salvage rate. Vessel thrombosis predicted inability to salvage the flap. 4 © 2015 The American Laryngological, Rhinological and Otological Society, Inc.

  12. Kyphoplasty for osteoporotic fractures with spinal canal compromise

    International Nuclear Information System (INIS)

    Gan Minfeng; Yang Huilin; Zou Jun; Wang Genlin; Mei Xin; Zhou Feng; Chen Liang; Jiang Weimin

    2010-01-01

    Objective: To explore the feasibility and clinical outcome of kyphoplasty in the treatment of osteoporotic fractures with canal compromise. Methods: A total of 16 patients with osteoporotic fractures with canal compromise without neurological deficit were attempted to be treated by kyphoplasty. During kyphoplasty, modified techniques including staged bone cement injection and dynamic fluoroscopic monitoring were used. Pain was measured using the self-reporting Visual Analogue pain Scale (VAS) preoperatively, postoperatively and in the final follow-up. Disability was measured using the Oswestry Disability questionnaire (ODI) preoperatively, postoperatively and in the final follow-up. The height of the compromised vertebral body, the kyphotic angle and the spinal canal compromise were measured preoperatively, postoperatively and in the final follow-up. Results: Operations were completed smoothly, with the exception of one patient with less cement leakage but without clinical symptom occurred. Relief of pain was achieved after kyphoplasty. The mean VAS score of these patients decreased from 8.1 ± 1.2 pre-operatively to 2.7 ± 0.6 post-operatively (P 0.05). In the final follow-up, the spinal canal compromise was (14.4 ± 3.1)%. Conclusion: Kyphoplasty is a relatively safe and effective method for the treatment of osteoporotic fractures with canal compromise without neurological deficit. (authors)

  13. A novel protease activity assay using a protease-responsive chaperone protein

    International Nuclear Information System (INIS)

    Sao, Kentaro; Murata, Masaharu; Fujisaki, Yuri; Umezaki, Kaori; Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki; Hashizume, Makoto

    2009-01-01

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  14. A novel protease activity assay using a protease-responsive chaperone protein

    Energy Technology Data Exchange (ETDEWEB)

    Sao, Kentaro [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Murata, Masaharu, E-mail: m-murata@dem.med.kyushu-u.ac.jp [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Fujisaki, Yuri; Umezaki, Kaori [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan); Mori, Takeshi; Niidome, Takuro; Katayama, Yoshiki [Graduate School of Systems Life Sciences, Kyushu University, 744 Motooka Nishi-ku, Fukuoka 819-0395 (Japan); Department of Applied Chemistry, Faculty of Engineering, Kyushu University, Nishi-ku Fukuoka 819-0395 (Japan); Center for Future Chemistry, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395 (Japan); Hashizume, Makoto [Department of Advanced Medical Initiatives, Faculty of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku Fukuoka 812-8582 (Japan)

    2009-06-05

    Protease activity assays are important for elucidating protease function and for developing new therapeutic agents. In this study, a novel turbidimetric method for determining the protease activity using a protease-responsive chaperone protein is described. For this purpose, a recombinant small heat-shock protein (sHSP) with an introduced Factor Xa protease recognition site was synthesized in bacteria. This recombinant mutant, FXa-HSP, exhibited chaperone-like activity at high temperatures in cell lysates. However, the chaperone-like activity of FXa-HSP decreased dramatically following treatment with Factor Xa. Protein precipitation was subsequently observed in the cell lysates. The reaction was Factor Xa concentration-dependent and was quantitatively suppressed by a specific inhibitor for Factor Xa. Protein aggregation was detected by a simple method based on turbidimetry. The results clearly demonstrate that this assay is an effective, easy-to-use method for determining protease activities without the requirement of labeling procedures and the use of radioisotopes.

  15. Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy.

    Science.gov (United States)

    Musante, Luca; Tataruch, Dorota; Gu, Dongfeng; Liu, Xinyu; Forsblom, Carol; Groop, Per-Henrik; Holthofer, Harry

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  16. Proteases and Protease Inhibitors of Urinary Extracellular Vesicles in Diabetic Nephropathy

    Directory of Open Access Journals (Sweden)

    Luca Musante

    2015-01-01

    Full Text Available Diabetic nephropathy (DN is one of the major complications of diabetes mellitus (DM, leads to chronic kidney disease (CKD, and, ultimately, is the main cause for end-stage kidney disease (ESKD. Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes.

  17. HIV protease drug resistance and its impact on inhibitor design.

    Science.gov (United States)

    Ala, P J; Rodgers, J D; Chang, C H

    1999-07-01

    The primary cause of resistance to the currently available HIV protease inhibitors is the accumulation of multiple mutations in the viral protease. So far more than 20 substitutions have been observed in the active site, dimer interface, surface loops and flaps of the homodimer. While many mutations reduce the protease's affinity for inhibitors, others appear to enhance its catalytic efficiency. This high degree of genetic flexibility has made the protease an elusive drug target. The design of the next generation of HIV protease inhibitors will be discussed in light of the current structural information.

  18. Pathophysiological significance and therapeutic applications of snake venom protease inhibitors.

    Science.gov (United States)

    Thakur, Rupamoni; Mukherjee, Ashis K

    2017-06-01

    Protease inhibitors are important constituents of snake venom and play important roles in the pathophysiology of snakebite. Recently, research on snake venom protease inhibitors has provided valuable information to decipher the molecular details of various biological processes and offer insight for the development of some therapeutically important molecules from snake venom. The process of blood coagulation and fibrinolysis, in addition to affecting platelet function, are well known as the major targets of several snake venom protease inhibitors. This review summarizes the structure-functional aspects of snake venom protease inhibitors that have been described to date. Because diverse biological functions have been demonstrated by protease inhibitors, a comparative overview of their pharmacological and pathophysiological properties is also highlighted. In addition, since most snake venom protease inhibitors are non-toxic on their own, this review evaluates the different roles of individual protease inhibitors that could lead to the identification of drug candidates and diagnostic molecules. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Microbial glycoproteomics

    DEFF Research Database (Denmark)

    Halim, Adnan; Anonsen, Jan Haug

    2017-01-01

    Mass spectrometry-based "-omics" technologies are important tools for global and detailed mapping of post-translational modifications. Protein glycosylation is an abundant and important post translational modification widespread throughout all domains of life. Characterization of glycoproteins...... and research in this area is rapidly accelerating. Here, we review recent developments in glycoproteomic technologies with a special focus on microbial protein glycosylation....

  20. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

  1. Microbial keratinases: industrial enzymes with waste management potential.

    Science.gov (United States)

    Verma, Amit; Singh, Hukum; Anwar, Shahbaz; Chattopadhyay, Anirudha; Tiwari, Kapil K; Kaur, Surinder; Dhilon, Gurpreet Singh

    2017-06-01

    Proteases are ubiquitous enzymes that occur in various biological systems ranging from microorganisms to higher organisms. Microbial proteases are largely utilized in various established industrial processes. Despite their numerous industrial applications, they are not efficient in hydrolysis of recalcitrant, protein-rich keratinous wastes which result in environmental pollution and health hazards. This paved the way for the search of keratinolytic microorganisms having the ability to hydrolyze "hard to degrade" keratinous wastes. This new class of proteases is known as "keratinases". Due to their specificity, keratinases have an advantage over normal proteases and have replaced them in many industrial applications, such as nematicidal agents, nitrogenous fertilizer production from keratinous waste, animal feed and biofuel production. Keratinases have also replaced the normal proteases in the leather industry and detergent additive application due to their better performance. They have also been proved efficient in prion protein degradation. Above all, one of the major hurdles of enzyme industrial applications (cost effective production) can be achieved by using keratinous waste biomass, such as chicken feathers and hairs as fermentation substrate. Use of these low cost waste materials serves dual purposes: to reduce the fermentation cost for enzyme production as well as reducing the environmental waste load. The advent of keratinases has given new direction for waste management with industrial applications giving rise to green technology for sustainable development.

  2. Luminometric method for screening retroviral protease inhibitors

    Czech Academy of Sciences Publication Activity Database

    Horáková, D.; Rumlová, Michaela; Pichová, Iva; Ruml, Tomáš

    2005-01-01

    Roč. 345, č. 1 (2005), s. 96-101 ISSN 0003-2697 R&D Projects: GA AV ČR(CZ) IAA4055304; GA MŠk(CZ) 1M0508; GA MŠk(CZ) 1M0520 Institutional research plan: CEZ:AV0Z40550506 Keywords : retroviral protease * inhibitors * luminescent assay Subject RIV: CE - Biochemistry Impact factor: 2.670, year: 2005

  3. Alchemy or Science? Compromising Archaeology in the Deep Sea

    Science.gov (United States)

    Adams, Jonathan

    2007-06-01

    In the torrid debate between archaeology and treasure hunting, compromise is often suggested as the pragmatic solution, especially for archaeology carried out either in deep water or beyond the constraints that commonly regulate such activities in territorial seas. Both the wisdom and the need for such compromise have even been advocated by some archaeologists, particularly in forums such as the internet and conferences. This paper argues that such a compromise is impossible, not in order to fuel confrontation but simply because of the nature of any academic discipline. We can define what archaeology is in terms of its aims, theories, methods and ethics, so combining it with an activity founded on opposing principles must transform it into something else. The way forward for archaeology in the deep sea does not lie in a contradictory realignment of archaeology’s goals but in collaborative research designed to mesh with emerging national and regional research and management plans.

  4. Compromise decision support problems for hierarchical design involving uncertainty

    Science.gov (United States)

    Vadde, S.; Allen, J. K.; Mistree, F.

    1994-08-01

    In this paper an extension to the traditional compromise Decision Support Problem (DSP) formulation is presented. Bayesian statistics is used in the formulation to model uncertainties associated with the information being used. In an earlier paper a compromise DSP that accounts for uncertainty using fuzzy set theory was introduced. The Bayesian Decision Support Problem is described in this paper. The method for hierarchical design is demonstrated by using this formulation to design a portal frame. The results are discussed and comparisons are made with those obtained using the fuzzy DSP. Finally, the efficacy of incorporating Bayesian statistics into the traditional compromise DSP formulation is discussed and some pending research issues are described. Our emphasis in this paper is on the method rather than the results per se.

  5. Role of Proteases in Chronic Obstructive Pulmonary Disease

    Directory of Open Access Journals (Sweden)

    Kailash C. Pandey

    2017-08-01

    Full Text Available Chronic obstructive pulmonary disease (COPD is generally associated with progressive destruction of airways and lung parenchyma. Various factors play an important role in the development and progression of COPD, like imbalance of proteases, environmental and genetic factors and oxidative stress. This review is specifically focused on the role of proteases and their imbalance in COPD. There are three classes (serine, mettalo, and cysteine of proteases involved in COPD. In serine proteases, neutrophil elastase, cathepsin G, and proteinase-3 are involved in destruction of alveolar tissue. Matrix-mettaloproteinase-9, 12, 13, plays an influential role in severity of COPD. Among cysteine proteases, caspase-3, caspases-8 and caspase-9 play an important role in controlling apoptosis. These proteases activities can be regulated by inhibitors like α-1-antitrypsin, neutrophil elastase inhibitor, and leukocyte protease inhibitor. Studies suggest that neutrophil elastase may be a therapeutic target for COPD, and specific inhibitor against this enzyme has potential role to control the disease. Current study suggests that Dipeptidyl Peptidase IV is a potential marker for COPD. Since the expression of proteases and its inhibitors play an important role in COPD pathogenesis, therefore, it is worth investigating the role of proteases and their regulation. Understanding the biochemical basis of COPD pathogenesis using advanced tools in protease biochemistry and aiming toward translational research from bench-to-bedside will have great impact to deal with this health problem.

  6. PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

    Directory of Open Access Journals (Sweden)

    R. Satheeskumar

    2013-10-01

    Full Text Available Partial purification and characterization of alkalophilic protease production from Pseudomonas aeruginosa was isolated from the gut of marine and coastal waters shrimp Penaeus monodon. The protease production was assayed in submerged fermentation to produce maximum protease activity (423 ± 0.09 U/ml. The enzyme was precipitated with ammonium sulphate and partially purified by ion exchange chromatography through DEAE Sephadex A-50 column. In 10th fraction showed maximum protease activity (734 ± 0.18 U/ml with increase in purification fold. The molecular weight of protease from Pseudomonas aeruginosa was recorded as 60 kDa. The stability of protease was tested at various pH and temperature; it showed maximum protease activity at pH-9 and temperature 50ºC. Among the various surfactants tested for enzyme stability, maximum activity was retained in poly ethylene glycol. The compatibility of protease enzyme with various commercial detergents; the enzyme retained maximum protease activity in tide. The results are indicated that all these properties make the bacterial proteases are most suitable for wide industrial applications.

  7. Understanding serine proteases implications on Leishmania spp lifecycle.

    Science.gov (United States)

    Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

    2018-01-01

    Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  8. Corruption of innate immunity by bacterial proteases.

    Science.gov (United States)

    Potempa, Jan; Pike, Robert N

    2009-01-01

    The innate immune system of the human body has developed numerous mechanisms to control endogenous and exogenous bacteria and thus prevent infections by these microorganisms. These mechanisms range from physical barriers such as the skin or mucosal epithelium to a sophisticated array of molecules and cells that function to suppress or prevent bacterial infection. Many bacteria express a variety of proteases, ranging from non-specific and powerful enzymes that degrade many proteins involved in innate immunity to proteases that are extremely precise and specific in their mode of action. Here we have assembled a comprehensive picture of how bacterial proteases affect the host's innate immune system to gain advantage and cause infection. This picture is far from being complete since the numbers of mechanisms utilized are as astonishing as they are diverse, ranging from degradation of molecules vital to innate immune mechanisms to subversion of the mechanisms to allow the bacterium to hide from the system or take advantage of it. It is vital that such mechanisms are elucidated to allow strategies to be developed to aid the innate immune system in controlling bacterial infections.

  9. Physically Compromised and Physically Talented Children in Northeastern Slovenia

    Directory of Open Access Journals (Sweden)

    Planinšec Jurij

    2012-12-01

    Full Text Available The aim of our research was to determine the share of physically compromised and physically talented children in northeastern Slovenia. The sample comprised 621 children aged nine to eleven years, among which there were 316 girls (M=10; SD=0.8 and 305 boys (M=10; SD=0.81. In order to assess their motor skills, seven different tests were used, mostly from Eurofit test battery, which covered explosive power, repetitive power balance, eye–hand coordination, speed of simple movements, whole body coordination, and endurance. The assessment was made for each physical fitness test separately. The cut-off points for determination of physically compromised and physically talented children were set at -1SD and +1SD, respectively. The results of all physical fitness tests showed that the share of physically compromised children exceeded ten percent for both genders. The largest number of boys and girls were physically compromised with regard to endurance and balance, respectively. On the other hand, boys proved to be most physically talented with regard to endurance, and girls with regard to explosive power. Gender differences were most obvious with regard to general endurance, as 21 per cent of the boys were physically compromised as opposed to 13 per cent of the girls. As for physical talent, we observed less gender-related differences. The results indicate increasing differences in physical fitness among children from northeastern Slovenia. The implementation of curricular and extracurricular sports activities should aim at reducing the number of physically compromised children. On the other hand, it would make sense to encourage physically talented children to get involved in organized forms of exercise.

  10. Roles of Melatonin in Fetal Programming in Compromised Pregnancies

    Science.gov (United States)

    Chen, Yu-Chieh; Sheen, Jiunn-Ming; Tiao, Miao-Meng; Tain, You-Lin; Huang, Li-Tung

    2013-01-01

    Compromised pregnancies such as those associated with gestational diabetes mellitus, intrauterine growth retardation, preeclampsia, maternal undernutrition, and maternal stress may negatively affect fetal development. Such pregnancies may induce oxidative stress to the fetus and alter fetal development through the epigenetic process that may affect development at a later stage. Melatonin is an oxidant scavenger that reverses oxidative stress during the prenatal period. Moreover, the role of melatonin in epigenetic modifications in the field of developmental programming has been studied extensively. Here, we describe the physiological function of melatonin in pregnancy and discuss the roles of melatonin in fetal programming in compromised pregnancies, focusing on its involvement in redox and epigenetic mechanisms. PMID:23466884

  11. Fuzzy compromise: An effective way to solve hierarchical design problems

    Science.gov (United States)

    Allen, J. K.; Krishnamachari, R. S.; Masetta, J.; Pearce, D.; Rigby, D.; Mistree, F.

    1990-01-01

    In this paper, we present a method for modeling design problems using a compromise decision support problem (DSP) incorporating the principles embodied in fuzzy set theory. Specifically, the fuzzy compromise decision support problem is used to study hierarchical design problems. This approach has the advantage that although the system modeled has an element of uncertainty associated with it, the solution obtained is crisp and precise. The efficacy of incorporating fuzzy sets into the solution process is discussed in the context of results obtained for a portal frame.

  12. MUTUAL CONCESSIONS - SPECIFIC ELEMENT OF THE COMPROMISE/TRANSACTION CONTRACT

    Directory of Open Access Journals (Sweden)

    Georgeta-Bianca Spîrchez

    2012-11-01

    Full Text Available Given the usefulness and practical importance of the compromise contract conclusion and of the amicably dispute resolution, within the business world, we aim to analyze, in what follows, the concrete means by which these kind of settlement are achieved. Two questions become legitimate in the context of concerns about mutual concessions which the parties make in a compromise contract. These questions are the following: “What are the mutual concessions? Do mutual concessions mean equivalent concessions?” and “How mutual concessions are required to complete a valid settlement? Is the requirement of mutual concessions grounded?”

  13. Mixing compatibilities of Aspergillus and American cockroach allergens with other high-protease fungal and insect extracts.

    Science.gov (United States)

    Grier, Thomas J; Hall, Dawn M; Duncan, Elizabeth A; Coyne, Terrance C

    2015-03-01

    Recent studies have shown that Alternaria and German cockroach allergens can be degraded by endogenous proteases from other insect and fungal extracts when combined for immunotherapy, but data supporting the compatibilities of other high-protease products in comparable mixtures have not been reported. To assess the stabilities and compatibilities of Aspergillus fumigatus and American cockroach allergens after mixing with protease-rich extracts from other insects or fungi at concentrations similar to those recommended for subcutaneous immunotherapy. Mixtures containing A fumigatus, American cockroach, and other fungal or insect extracts were evaluated by quantitative (enzyme-linked immunosorbent assays) and qualitative (immunoblotting) methods. Test mixtures and control samples at 10% to 50% glycerin concentrations were analyzed after storage for up to 12 months at 2°C to 8°C. Moderate to high recoveries of Aspergillus extract activities were retained in control samples and extract mixtures under all conditions examined. American cockroach extract controls were partly degraded at 10% to 25% glycerin, and cockroach allergen compatibilities were decreased significantly in mixtures with several fungal extracts at 25% glycerin. Mixing with other insects did not compromise the stability of American cockroach allergens at 25% to 50% glycerin. Aspergillus extracts exhibited favorable stabilities after mixing with other high-protease products. American cockroach extract potencies were unstable in less than 50% glycerin, even in the absence of other protease-containing allergens, and were destabilized in mixtures with several fungal extracts. Addition of fungal and insect extracts to separate treatment vials or preparation of fungal-insect mixtures at elevated glycerin concentrations might be necessary to produce compatible patient formulations for allergen immunotherapy injections. Copyright © 2015 American College of Allergy, Asthma & Immunology. Published by Elsevier

  14. Microbial xanthophylls.

    Science.gov (United States)

    Bhosale, Prakash; Bernstein, Paul S

    2005-09-01

    Xanthophylls are oxygenated carotenoids abundant in the human food supply. Lutein, zeaxanthin, and cryptoxanthin are major xanthophyll carotenoids in human plasma. The consumption of these xanthophylls is directly associated with reduction in the risk of cancers, cardiovascular disease, age-related macular degeneration, and cataract formation. Canthaxanthin and astaxanthin also have considerable importance in aquaculture for salmonid and crustacean pigmentation, and are of commercial interest for the pharmaceutical and food industries. Chemical synthesis is a major source for the heavy demand of xanthophylls in the consumer market; however, microbial producers also have potential as commercial sources. In this review, we discuss the biosynthesis, commercial utility, and major microbial sources of xanthophylls. We also present a critical review of current research and technologies involved in promoting microbes as potential commercial sources for mass production.

  15. Tax Administration: IRS Should Evaluate the Changes to Its Offer in Compromise Program

    National Research Council Canada - National Science Library

    2002-01-01

    ...) Offer in Compromise (OIC) Program. An offer in compromise is an agreement between a taxpayer and the IRS to settle or compromise the taxpayer's tax liability for less than the full amount owed...

  16. Studies On Optimization Of Protease Production Using Bacterial Isolate Clri Strain 5468 And Its Application In Dehairing And Hydrolysis Of Tannery Fleshings Solid Waste Management

    Directory of Open Access Journals (Sweden)

    Vimala Devi Seenivasagham

    2015-08-01

    Full Text Available The strain which produces protease was originally isolated characterized in Biotechnology laboratory at CLRI and was maintained. The microorganism was growned on several proteolytic media and the maximum activity was observed. The characterization of enzyme was analysed for different pH temperature size of inoculum inhibitors age of the culture. Then the enzyme was observed for the unhairing of skin and the disadvantage in chemical treatment was studied. The conformation of unhairing was studied using histology studies. The tannery waste solid fleshings as it is cannot be directly disposed off to the environment. It was treated with the microbial proteases. The hydrolysis of waste was done using proteases. The solid waste was converted to protien fat and the salt matter. Future work is to optimize the cheap media for the production of the enzyme for large scale applications in various industries.

  17. A theoretical reassessment of microbial maintenance and implications for microbial ecology modeling.

    Science.gov (United States)

    Wang, Gangsheng; Post, Wilfred M

    2012-09-01

    We attempted to reconcile three microbial maintenance models (Herbert, Pirt, and Compromise) through a theoretical reassessment. We provided a rigorous proof that the true growth yield coefficient (Y(G)) is the ratio of the specific maintenance rate (a in Herbert) to the maintenance coefficient (m in Pirt). Other findings from this study include: (1) the Compromise model is identical to the Herbert for computing microbial growth and substrate consumption, but it expresses the dependence of maintenance on both microbial biomass and substrate; (2) the maximum specific growth rate in the Herbert (μ(max,H)) is higher than those in the other two models (μ(max,P) and μ(max,C)), and the difference is the physiological maintenance factor (m(q) = a); and (3) the overall maintenance coefficient (m(T)) is more sensitive to m(q) than to the specific growth rate (μ(G)) and Y(G). Our critical reassessment of microbial maintenance provides a new approach for quantifying some important components in soil microbial ecology models. © This article is a US government work and is in the public domain in the USA.

  18. 47 CFR 1.1915 - Exploration of compromise.

    Science.gov (United States)

    2010-10-01

    ... justification of the offer and addressing the bases for compromise at 31 CFR 902.2. Debtors will provide full.... Unless otherwise provided by law, when the principal balance of a debt, exclusive of interest, penalties... evaluate an offer, using the factors set forth in 31 CFR 902.2 and, as appropriate, refer the offer with...

  19. Pulmonary, neurological and hepatic compromise in paragonimiasis: case report

    International Nuclear Information System (INIS)

    Uriza Carrasco, Alfonso Jose; Cuervo Valencia, Catalina; Valencia M, Andres Mauricio; Echeverri Toro, Lina Maria

    2011-01-01

    Paragonimiasis is a parasitic infectious disease, frequently with pulmonary abnormalities, although erratic parasite migration can compromise other organs. this disease is endemic in areas of southeast Asia, but it has been described in other tropical and subtropical regions. In this article, we report a case of indigenous patient with pulmonary abnormalities by Paragonimiasis, neurologic and hepatic abnormalities too.

  20. Adolescent Health-Compromising Behaviors: Motivating School Counselors.

    Science.gov (United States)

    Nagel, Liza; Scherer, David G.; Lee, William

    2000-01-01

    Investigated middle and high school counselors' perceptions of adolescent health-compromising behaviors and motivations to intervene. Data from a survey based on protection motivation theory showed differences in counselors' perceptions of the severity of risk-taking behaviors. Perceptions were highly correlated with intentions to seek out…

  1. Secretion of salivary statherin is compromised in uncontrolled diabetic patients

    Directory of Open Access Journals (Sweden)

    Masahiro Izumi

    2015-06-01

    Conclusions and general significance: The results show that synthesis and secretion of statherin is reduced in diabetics and this reduction is salivary gland specific. As compromised salivary statherin secretion leads to increased oral health risk, this study indicates that routine oral health assessment of these patients is warranted.

  2. Whatever It Takes: Health Compromising Behaviors in Female Athletes

    Science.gov (United States)

    Waldron, Jennifer J.; Krane, Vikki

    2005-01-01

    The power and performance model of sport stresses a sport ethic of doing "whatever it takes" to win (Coakley, 2004). Uncritical acceptance of this model may lead to various health-compromising behaviors. Employing achievement goal theory, we examine why female athletes may adopt the power and performance approach. An ego motivational climate and a…

  3. 14 CFR 1261.414 - Compromise of claims.

    Science.gov (United States)

    2010-01-01

    ... potential income; (3) Inheritance prospects; (4) The possibility that assets have been concealed or... compromised. The practical benefits of vigorous collection of a small claim may include a demonstration to... requiring a waiver of the tax-loss-carry-back rights of the debtor. (i) Joint and several liability. When...

  4. The medically compromised patient: Are dental implants a feasible option?

    Science.gov (United States)

    Vissink, A; Spijkervet, Fkl; Raghoebar, G M

    2018-03-01

    In healthy subjects, dental implants have evolved to be a common therapy to solve problems related to stability and retention of dentures as well as to replace failing teeth. Although dental implants are applied in medically compromised patients, it is often not well known whether this therapy is also feasible in these patients, whether the risk of implant failure and developing peri-implantitis is increased, and what specific preventive measures, if any, have to be taken when applying dental implants in these patients. Generally speaking, as was the conclusion by the leading review of Diz, Scully, and Sanz on placement of dental implants in medically compromised patients (J Dent, 41, 2013, 195), in a few disorders implant survival may be lower, and the risk of a compromised peri-implant health and its related complications be greater, but the degree of systemic disease control outweighs the nature of the disorder rather than the risk accompanying dental implant treatment. So, as dental implant treatment is accompanied by significant functional benefits and improved oral health-related quality of life, dental implant therapy is a feasible treatment in almost any medically compromised patient when the required preventive measures are taken and follow-up care is at a high level. © 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd. All rights reserved.

  5. 45 CFR 1177.12 - Compromise, suspension and termination.

    Science.gov (United States)

    2010-10-01

    ... 45 Public Welfare 3 2010-10-01 2010-10-01 false Compromise, suspension and termination. 1177.12 Section 1177.12 Public Welfare Regulations Relating to Public Welfare (Continued) NATIONAL FOUNDATION ON...) debts arising from GAO audit exceptions. ...

  6. The compromised gut in the neonate : Diagnostic and clinical aspects

    NARCIS (Netherlands)

    Schurink, Maarten

    2016-01-01

    Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease predominantly occurring in preterm infants, causing significant patient morbidity and mortality. It is characterized by loss of bowel wall integrity due to ischemia (lack of oxygen due to a compromised blood flow) and

  7. Integer goal programming approach for finding a compromise ...

    African Journals Online (AJOL)

    In second model the cost and time spent on repairing the components are considered as two different objectives. Selective maintenance operation is used to select the repairable components and a multi-objective goal programming algorithm is proposed to obtain compromise selection of repairable components for the two ...

  8. 31 CFR 902.2 - Bases for compromise.

    Science.gov (United States)

    2010-07-01

    ... 31 Money and Finance: Treasury 3 2010-07-01 2010-07-01 false Bases for compromise. 902.2 Section 902.2 Money and Finance: Treasury Regulations Relating to Money and Finance (Continued) FEDERAL CLAIMS... necessary to further an enforcement principle, such as the Government's willingness to pursue aggressively...

  9. Erwinia carotovora extracellular proteases : characterization and role in soft rot

    OpenAIRE

    Kyöstiö, Sirkka R. M.

    1990-01-01

    Erwinia carotovora subsp. carotovora (Ecc) strain EC14, a Gram-negative bacterium, causes soft rot on several crops, including potato. Maceration of potato tuber tissue is caused by secreted pectolytic enzymes. Other cell-degrading enzymes may also have roles in pathogenesis, including cellulases, phospholipases, and protease(s). The objectives of this research were to (1) characterize Ecc extracellular protease (Prt) and (2) elucidate its role in potato soft rot. A gene enc...

  10. Economic Methods of Ginger Protease'sextraction and Purification

    Science.gov (United States)

    Qiao, Yuanyuan; Tong, Junfeng; Wei, Siqing; Du, Xinyong; Tang, Xiaozhen

    This article reports the ginger protease extraction and purification methods from fresh ginger rhizome. As to ginger protease extraction, we adapt the steps of organic solvent dissolving, ammonium sulfate depositing and freeze-drying, and this method can attain crude enzyme powder 0.6% weight of fresh ginger rhizome. The purification part in this study includes two steps: cellulose ion exchange (DEAE-52) and SP-Sephadex 50 chromatography, which can purify crude ginger protease through ion and molecular weight differences respectively.

  11. Vatican is lone opponent of world conference's compromises on abortion.

    Science.gov (United States)

    1994-09-07

    Three years in the making, the draft program of action of the 1994 International Conference on Population and Development sets nonbinding policy guidelines to contain the world's population at 7.27 billion in 2015. Although the Vatican was pleased to see Pakistan put forward a compromise formula developed to appease Catholic and Muslim objectors of abortion, the Church was unprepared to accept the compromise immediately and requested further discussion. The Vatican's rejection drew a strong chorus of vocal disapproval from other conference delegates. Even Iran accepted the draft as a "perfect text," while Sweden grudgingly accepted it as a "rock-bottom compromise." With no Catholic countries objecting to the compromise, the Vatican stood alone in its refusal to compromise with the rest of the world's leaders and peoples. Germany, speaking for the European Union, warned that enough concessions had already been made. The rationale for Vatican opposition was unclear since the section explicitly rejects abortion as a means of family planning and urges countries to minimize both the incidence of unsafe abortion and abortion overall by improving family planning. Prevention of unwanted pregnancies must be given highest priority and women should have ready access to compassionate counselling, with abortion never promoted as a means of family planning. Moreover, there is no longer a reference to sexual health education, a plea to governments to review their laws and policies on abortion, and a call to consider women's health rather than relying upon criminal codes and punitive measures. Participants said the Vatican objected to a phrase stating that abortions, where legal, should be safe, while the Church representative argued that any suggestion that abortion is safe contradicts church doctrine on the sanctity of life.

  12. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins.

    Science.gov (United States)

    Sarpong, Kwabena; Bose, Ron

    2017-03-15

    A major challenge in attaching fluorophores or other handles to proteins is the availability of a site-specific labeling strategy that provides stoichiometric modification without compromising protein integrity. We developed a simple approach that combines TEV protease cleavage, sortase modification and affinity purification to N-terminally label proteins. To achieve stoichiometrically-labeled protein, we included a short affinity tag in the fluorophore-containing peptide for post-labeling purification of the modified protein. This strategy can be easily applied to any recombinant protein with a TEV site and we demonstrate this on Epidermal Growth Factor Receptor (EGFR) and Membrane Scaffold Protein (MSP) constructs. Copyright © 2017 Elsevier Inc. All rights reserved.

  13. Retaining in-gel zymographic activity of cysteine proteases via a cysteine-supplemented running buffer.

    Science.gov (United States)

    Vootukuri Reddy, Sreekanth; Philpott, Mike P; Trigiante, Giuseppe

    2016-10-01

    Zymography is a powerful technique to separate and identify different enzymatic activities on a standard acrylamide gel. For oxidation prone enzymes such as cysteine proteases however, the oxidizing species generated by electrolysis of the gel running buffer may result in partial or complete inactivation, thus compromising the final readout. This can be only partially remedied by subsequent treatment of the gel with reducing agents. We demonstrate the generation of reactive oxidizing species during electrophoresis and discovered that supplementation of the gel running buffer with a minimum of 5 mM cysteine prevents enzyme inactivation and allows retention of proteolytic activity as measured by zymography on model substrate N α-benzoyl-l-arginine p-nitroanilide, without at the same time altering the mobilities of the gel proteins. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  14. Indispensable Role of Proteases in Plant Innate Immunity.

    Science.gov (United States)

    Balakireva, Anastasia V; Zamyatnin, Andrey A

    2018-02-23

    Plant defense is achieved mainly through the induction of microbe-associated molecular patterns (MAMP)-triggered immunity (MTI), effector-triggered immunity (ETI), systemic acquired resistance (SAR), induced systemic resistance (ISR), and RNA silencing. Plant immunity is a highly complex phenomenon with its own unique features that have emerged as a result of the arms race between plants and pathogens. However, the regulation of these processes is the same for all living organisms, including plants, and is controlled by proteases. Different families of plant proteases are involved in every type of immunity: some of the proteases that are covered in this review participate in MTI, affecting stomatal closure and callose deposition. A large number of proteases act in the apoplast, contributing to ETI by managing extracellular defense. A vast majority of the endogenous proteases discussed in this review are associated with the programmed cell death (PCD) of the infected cells and exhibit caspase-like activities. The synthesis of signal molecules, such as salicylic acid, jasmonic acid, and ethylene, and their signaling pathways, are regulated by endogenous proteases that affect the induction of pathogenesis-related genes and SAR or ISR establishment. A number of proteases are associated with herbivore defense. In this review, we summarize the data concerning identified plant endogenous proteases, their effect on plant-pathogen interactions, their subcellular localization, and their functional properties, if available, and we attribute a role in the different types and stages of innate immunity for each of the proteases covered.

  15. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

    Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben B

    Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned with and w......Cysteine Proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins during germination. Several Cysteine proteases have been identified in barley. One of the key enzymes, Hordeum vulgare endoprotease B2 (HvEPB2) was cloned...

  16. Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

    Directory of Open Access Journals (Sweden)

    Tanveer Majeed

    2013-01-01

    Full Text Available A putative protease gene (aprE from the thermophilic bacterium Coprothermobacter proteolyticus was cloned and expressed in Bacillus subtilis. The enzyme was determined to be a serine protease based on inhibition by PMSF. Biochemical characterization demonstrated that the enzyme had optimal activity under alkaline conditions (pH 8–10. In addition, the enzyme had an elevated optimum temperature (60°C. The protease was also stable in the presence of many surfactants and oxidant. Thus, the C. proteolyticus protease has potential applications in industries such as the detergent market.

  17. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Directory of Open Access Journals (Sweden)

    Suk Ho Choi

    2011-09-01

    Full Text Available Objectives : This study was undertaken to identify fibrinolytic proteases from Gloydius blomhoffii siniticus venom and to characterize a major fibrinolytic protease purified from the venom. Methods: The venom was subjected to chromatography using columns of Q-Sepharose and Sephadex G-75. The molecular weights of fibrinolytic proteases showing fibrinolytic zone in fibrin plate assay were determined in SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel electrophoresis The effects of inhibitors and metal ions on fibrinolytic protease and the proteolysis patterns of fibrinogen, gelatin, and bovine serum albumin were investigated. Results : 1 The fibrinolytic fractions of the three peaks isolated from Gloydius blomhoffii siniticus venom contained two polypeptides of 46 and 59 kDa and three polypeptides of 32, 18, and 15 kDa and a major polypeptide of 54 kDa, respectively. 2 The fibrinolytic activity of the purified protease of 54 kDA was inhibited by metal chelators, such as EDTA, EGTA, and 1,10-phenanthroline, and disulfhydryl-reducing compounds, such as dithiothreitol and cysteine. 3 Calcium chloride promoted the fibrinolytic activity of the protease, but mercuric chloride and cobalt(II chloride inhibited it. 4 The fibrinolytic protease cleaved preferentially A-chain and slowly B-chain of fibrinogen. It also hydrolyzed gelatin but not bovine serum albumin. Conclusions: The Gloydius blomhoffii siniticus venom contained more than three fibrinolytic proteases. The major fibrinolytic protease was a metalloprotease which hydrolyzed both fibrinogen and gelatin, but not bovine serum albumin.

  18. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    International Nuclear Information System (INIS)

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-01-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  19. Intracellular alkaline proteases produced by thermoacidophiles: detection of protease heterogeneity by gelatin zymography and polymerase chain reaction (PCR)

    Energy Technology Data Exchange (ETDEWEB)

    Kocab, S.; Erdem, B. [Middle East Technical University, Ankara (Turkey). Dept. of Biological Sciences

    2002-08-01

    In this study 24 thermoacidophilic archeal and bacterial strains isolated from hot-springs and hot-soils were screened for their ability to produce intracellular alkaline proteases. The protease activities of the strains, based on azocasein hydrolysis, showed a variation from 0.6 to 5.1 U. The cell extracts of three most potent producers were further examined and it was found that their proteases exhibited maximum activity at 60-70{sup o}C and showed a pH optimum over a range of pH 7.0-8.5. Gelatin zymography revealed that two of the selected archeal strains produced multiple active SDS-resistant proteases. On the other hand, PCR amplification of alkaline serine protease gene sequences of total DNA from all isolates yielded four distinct amplification fragments of 650, 450, 400 and 300 bp, which might have been derived from different serine protease genes. (author)

  20. Enzymatic activity of proteases and its isoenzymes in fermentation process in cultivars of cocoa (Theobroma cacao L. produced in southern Bahia, Brazil

    Directory of Open Access Journals (Sweden)

    Luciane Santos SOUSA

    Full Text Available Abstract The fermentation of cocoa seeds envolves microbial processes and the action of enzymes. To identify the possible differences in the cocoa fermentation process, with regards to proteolysis, this study has the objective of determining protease activity (under predetermined conditions and its isoenzymes in two cocoa cultivars (PH-16 and HRT-1188 in different cocoa fermentation times, in addition to establishing the microbial load (molds and yeasts and aerobic mesophilic. Protease and its isoenzymes were extracted and partially purified and the enzymatic activities determined by spectrophotometry. The results showed that the proteases activity was higher at 66h of fermentation for both cultivars. When the isoenzymes activity was evaluated, the results demonstrated similar activity behavior for both cultivars, with regards to the isoenzymes aminopeptidase and carboxypeptidase, although the behavior of the endoprotease isoenzyme activity proved to be a little different for TSH-1188 cultivar. Concerning microbiological analyses, the results indicate that the period after molds and yeast counting reduction is consistent with the period of protease activity increase.

  1. Microbial effects

    International Nuclear Information System (INIS)

    Sharpe, V.J.

    1985-10-01

    The long term safety and integrity of radioactive waste disposal sites proposed for use by Ontario Hydro may be affected by the release of radioactive gases. Microbes mediate the primary pathways of waste degradation and hence an assessment of their potential to produce gaseous end products from the breakdown of low level waste was performed. Due to a number of unknown variables, assumptions were made regarding environmental and waste conditions that controlled microbial activity; however, it was concluded that 14 C and 3 H would be produced, albeit over a long time scale of about 1500 years for 14 C in the worst case situation

  2. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    Science.gov (United States)

    Panigrahi, Rashmi; Arutyunova, Elena; Panwar, Pankaj; Gimpl, Katharina; Keller, Sandro; Lemieux, M Joanne

    2016-03-29

    Denaturant-induced unfolding of helical membrane proteins provides insights into their mechanism of folding and domain organization, which take place in the chemically heterogeneous, anisotropic environment of a lipid membrane. Rhomboid proteases are intramembrane proteases that play key roles in various diseases. Crystal structures have revealed a compact helical bundle with a buried active site, which requires conformational changes for the cleavage of transmembrane substrates. A dimeric form of the rhomboid protease has been shown to be important for activity. In this study, we examine the mechanism of refolding for two distinct rhomboids to gain insight into their secondary structure-activity relationships. Although helicity is largely abolished in the unfolded states of both proteins, unfolding is completely reversible for HiGlpG but only partially reversible for PsAarA. Refolding of both proteins results in reassociation of the dimer, with a 90% regain of catalytic activity for HiGlpG but only a 70% regain for PsAarA. For both proteins, a broad, gradual transition from the native, folded state to the denatured, partly unfolded state was revealed with the aid of circular dichroism spectroscopy as a function of denaturant concentration, thus arguing against a classical two-state model as found for many globular soluble proteins. Thermal denaturation has irreversible destabilizing effects on both proteins, yet reveals important functional details regarding substrate accessibility to the buried active site. This concerted biophysical and functional analysis demonstrates that HiGlpG, with a simple six-transmembrane-segment organization, is more robust than PsAarA, which has seven predicted transmembrane segments, thus rendering HiGlpG amenable to in vitro studies of membrane-protein folding. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

  3. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    Energy Technology Data Exchange (ETDEWEB)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Roszak, Aleksander W. [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom); Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel, E-mail: daniel.walker@glasgow.ac.uk [University of Glasgow, Glasgow G12 8QQ, Scotland (United Kingdom)

    2015-06-30

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group.

  4. Structure of protease-cleaved Escherichia coli α-2-macroglobulin reveals a putative mechanism of conformational activation for protease entrapment

    International Nuclear Information System (INIS)

    Fyfe, Cameron D.; Grinter, Rhys; Josts, Inokentijs; Mosbahi, Khedidja; Roszak, Aleksander W.; Cogdell, Richard J.; Wall, Daniel M.; Burchmore, Richard J. S.; Byron, Olwyn; Walker, Daniel

    2015-01-01

    The X-ray structure of protease-cleaved E. coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. Bacterial α-2-macroglobulins have been suggested to function in defence as broad-spectrum inhibitors of host proteases that breach the outer membrane. Here, the X-ray structure of protease-cleaved Escherichia coli α-2-macroglobulin is described, which reveals a putative mechanism of activation and conformational change essential for protease inhibition. In this competitive mechanism, protease cleavage of the bait-region domain results in the untethering of an intrinsically disordered region of this domain which disrupts native interdomain interactions that maintain E. coli α-2-macroglobulin in the inactivated form. The resulting global conformational change results in entrapment of the protease and activation of the thioester bond that covalently links to the attacking protease. Owing to the similarity in structure and domain architecture of Escherichia coli α-2-macroglobulin and human α-2-macroglobulin, this protease-activation mechanism is likely to operate across the diverse members of this group

  5. Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors.

    Science.gov (United States)

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

    Dengue virus serotypes 1-4 (DENV1-4) are mosquito-borne human pathogens of global significance causing ~390 million cases annually worldwide. The virus infections cause in general a self-limiting disease, known as dengue fever, but occasionally also more severe forms, especially during secondary infections, dengue hemorrhagic fever and dengue shock syndrome causing ~25,000 deaths annually. The DENV genome contains a single-strand positive sense RNA, approximately 11 kb in length. The 5'-end has a type I cap structure. The 3'-end has no poly(A) tail. The viral RNA has a single long open reading frame that is translated by the host translational machinery to yield a polyprotein precursor. Processing of the polyprotein precursor occurs co-translationally by cellular proteases and posttranslationally by the viral serine protease in the endoplasmic reticulum (ER) to yield three structural proteins (capsid (C), precursor membrane (prM), and envelope (E) and seven nonstructural (NS) proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5). The active viral protease consists of both NS2B, an integral membrane protein in the ER, and the N-terminal part of NS3 (180 amino acid residues) that contains the trypsin-like serine protease domain having a catalytic triad of H51, D75, and S135. The C-terminal part of NS3, ~170-618 amino acid residues, encodes an NTPase/RNA helicase and 5'-RNA triphosphatase activities; the latter enzyme is required for the first step in 5'-capping. The cleavage sites of the polyprotein by the viral protease consist of two basic amino acid residues such as KR, RR, or QR, followed by short chain amino acid residues, G, S, or T. Since the cleavage of the polyprotein by the viral protease is absolutely required for assembly of the viral replicase, blockage of NS2B/NS3pro activity provides an effective means for designing dengue virus (DENV) small-molecule therapeutics. Here we describe the screening of small-molecule inhibitors against DENV2 protease.

  6. Complexity of cancer protease biology: Cathepsin K expression and function in cancer progression

    NARCIS (Netherlands)

    Verbovšek, Urška; van Noorden, Cornelis J. F.; Lah, Tamara T.

    2015-01-01

    Proteases, including lysosomal cathepsins, are functionally involved in many processes in cancer progression from its initiation to invasion and metastatic spread. Only recently, cathepsin K (CatK), the cysteine protease originally reported as a collagenolytic protease produced by osteoclasts,

  7. HIV-1 protease-substrate coevolution in nelfinavir resistance.

    Science.gov (United States)

    Kolli, Madhavi; Ozen, Ayşegül; Kurt-Yilmaz, Nese; Schiffer, Celia A

    2014-07-01

    Resistance to various human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. The virus accumulates mutations within the protease (PR) that render the PIs less potent. Occasionally, Gag sequences also coevolve with mutations at PR cleavage sites contributing to drug resistance. In this study, we investigated the structural basis of coevolution of the p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations by determining crystal structures of wild-type and NFV-resistant HIV-1 protease in complex with p1-p6 substrate peptide variants with L449F and/or S451N. Alterations of residue 30's interaction with the substrate are compensated by the coevolving L449F and S451N cleavage site mutations. This interdependency in the PR-p1-p6 interactions enhances intermolecular contacts and reinforces the overall fit of the substrate within the substrate envelope, likely enabling coevolution to sustain substrate recognition and cleavage in the presence of PR resistance mutations. Resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors challenges the effectiveness of therapies in treating HIV-1-infected individuals and AIDS patients. Mutations in HIV-1 protease selected under the pressure of protease inhibitors render the inhibitors less potent. Occasionally, Gag sequences also mutate and coevolve with protease, contributing to maintenance of viral fitness and to drug resistance. In this study, we investigated the structural basis of coevolution at the Gag p1-p6 cleavage site with the nelfinavir (NFV) resistance D30N/N88D protease mutations. Our structural analysis reveals the interdependency of protease-substrate interactions and how coevolution may restore substrate recognition and cleavage in the presence of protease drug resistance mutations. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  8. Optimization of Protease Production by Psychrotrophic Rheinheimera sp. with Response Surface Methodology

    Directory of Open Access Journals (Sweden)

    Mrayam Mahjoubin-Tehran

    2016-10-01

    Full Text Available Background and Objectives: Psychrotrophic bacteria can produce enzymes at low temperatures; this provides a wide biotechnological potential, and offers numerous economical advantages over the use of mesophilic bacteria. In this study, extracellular protease production by psychrotrophic Rheinheimera sp. (KM459533 was optimized by the response surface methodology.Materials and Methods: The culture medium was tryptic soy broth containing 1% (w v -1 skim milk. First, the effects of variables were independently evaluated on the microbial growth and protease production by one-factor-at-a-time method within the following ranges: incubation time 24-120 h, temperature 15-37°C, pH 6- 11, skim milk concentration 0-2% (w v -1 , and inoculum size 0.5-3% (v v -1 . The combinational effects of the four major variable including temperature, pH, skim milk concentration, and inoculum size were then evaluated within 96 h using response surface methodology through 27 experiments.Results and Conclusion: In one-factor-at-a-time method, high cell density was detected at 72h, 20°C, pH 7, skim milk 2% (w v -1 , and inoculum size 3% (v v -1 , and maximum enzyme production (533.74 Uml-1 was achieved at 96h, 20°C, pH 9, skim milk 1% (w v -1 , and inoculum size 3% (v v -1 . The response surface methodology study showed that pH is the most effective factor in enzyme production, and among the other variables, only temperature had significant interaction with pH and inoculum size. The determination coefficient (R2 =0.9544 and non-significant lack of fit demonstrated correlation between the experimental and predicted values. The optimal conditions predicted by the response surface methodology for protease production were defined as: 22C, pH 8.5, skim milk 1.1% (w v -1 , and inoculum size 4% (v v -1 . Protease production under these conditions reached to 567.19 Uml-1 . The use of response surface methodology in this study increased protease production by eight times as

  9. Securing Single Points of Compromise (SPoC)

    Energy Technology Data Exchange (ETDEWEB)

    Belangia, David Warren [Los Alamos National Lab. (LANL), Los Alamos, NM (United States)

    2015-06-25

    Securing the Single Points of Compromise that provide central services to the institution’s environment is paramount to success when trying to protect the business. (Fisk, 2014) Time Based Security mandates protection (erecting and ensuring effective controls) that last longer than the time to detect and react to a compromise. When enterprise protections fail, providing additional layered controls for these central services provides more time to detect and react. While guidance is readily available for securing the individual critical asset, protecting these assets as a group is not often discussed. Using best business practices to protect these resources as individual assets while leveraging holistic defenses for the group increases the opportunity to maximize protection time, allowing detection and reaction time for the SPoCs that is commensurate with the inherent risk of these centralized services.

  10. Roles of Melatonin in Fetal Programming in Compromised Pregnancies

    Directory of Open Access Journals (Sweden)

    Yu-Chieh Chen

    2013-03-01

    Full Text Available Compromised pregnancies such as those associated with gestational diabetes mellitus, intrauterine growth retardation, preeclampsia, maternal undernutrition, and maternal stress may negatively affect fetal development. Such pregnancies may induce oxidative stress to the fetus and alter fetal development through the epigenetic process that may affect development at a later stage. Melatonin is an oxidant scavenger that reverses oxidative stress during the prenatal period. Moreover, the role of melatonin in epigenetic modifications in the field of developmental programming has been studied extensively. Here, we describe the physiological function of melatonin in pregnancy and discuss the roles of melatonin in fetal programming in compromised pregnancies, focusing on its involvement in redox and epigenetic mechanisms.

  11. Creating clones, kids & chimera: liberal democratic compromise at the crossroads.

    Science.gov (United States)

    Adams, Nathan A

    2004-01-01

    The objective of this article is to find middle ground between the supporters and opponents of biotechnology by perpetuating the existing legal compromise pertaining to the complete range of health and welfare doctrines relevant to the biotechnological industry. The author aspires neither to add to nor detract from this liberal democratic consensus, but to preserve its constitutive balance between positivism and natural law and over-regulation and under-regulation in the hopes of stabilizing new political fault lines developing around the few biotechnological innovations already grabbing headlines. The most feasible solution is to extend the existing liberal democratic compromise with respect to equal protection, reproductive rights, the First Amendment, human subject experimentation, patent law, and parental rights. This includes banning or monopolizing certain biotechnologies and extending substantive special respect to the ex vivo living human embryo. Biotechnology must not be left to regulate itself.

  12. A Compromise Programming Model for Highway Maintenance Resources Allocation Problem

    Directory of Open Access Journals (Sweden)

    Hui Xiong

    2012-01-01

    Full Text Available This paper formulates a bilevel compromise programming model for allocating resources between pavement and bridge deck maintenances. The first level of the model aims to solve the resource allocation problems for pavement management and bridge deck maintenance, without considering resource sharing between them. At the second level, the model uses the results from the first step as an input and generates the final solution to the resource-sharing problem. To solve the model, the paper applies genetic algorithms to search for the optimal solution. We use a combination of two digits to represent different maintenance types. Results of numerical examples show that the conditions of both pavements and bridge decks are improved significantly by applying compromise programming, rather than conventional methods. Resources are also utilized more efficiently when the proposed method is applied.

  13. Would Enforcing Competition Law Compromise Industry Policy Objectives?

    OpenAIRE

    Evenett, Simon J.

    2005-01-01

    One recurring concern in the debate over the efficacy of enacting competition laws in developing countries is that its enforcement may compromise important industrial policy goals. This concern has been raised in regional fora and in multilateral organizations such as the World Trade Organization, where officials have considered the pros and cons of including competition provisions in international trade agreements. However, the concern is broader and often national debates over the merits of...

  14. Mechanisms and cellular functions of intramembrane proteases.

    Science.gov (United States)

    Urban, Siniša

    2013-12-01

    The turn of the millennium coincided with the branding of a fundamentally different class of enzyme - proteases that reside immersed inside the membrane. This new field was the convergence of completely separate lines of research focused on cholesterol homeostasis, Alzheimer's disease, and developmental genetics. None intended their ultimate path, but soon became a richly-integrated fabric for an entirely new field: regulated intramembrane proteolysis. Our aim in this Special Issue is to focus on the ancient and nearly ubiquitous enzymes that catalyze this unexpected yet important reaction. The pace of progress has been dramatic, resulting in a rapidly-expanding universe of known cellular functions, and a paradigm shift in the biochemical understanding of these once heretical enzymes. More recently, the first therapeutic successes have been attained by targeting an intramembrane protease. We consider these advances and identify oncoming opportunities in four parts: growing spectra of cellular roles, insights into biochemical mechanisms, therapeutic strategies, and newly-emerging topics. Recent studies also expose challenges for the future, including non-linear relationships between substrate identification and physiological functions, and the need for potent and specific, not broad-class, inhibitors. © 2013.

  15. Compromised Agency: The Case of BabyLegs

    Directory of Open Access Journals (Sweden)

    Max Liboiron

    2017-09-01

    Full Text Available The concept of agency is ubiquitous in STS, particularly regarding cases of alternative ways of knowing and doing science such as civic, citizen, and feminist sciences, among others. Yet the focus on agency often glosses over the constraints placed on agents, particularly within asymmetrical power relations. This article follows the case of BabyLegs, a do-it-yourself monitoring tool for marine microplastic pollution, and the attempt to keep the technology open source within an intellectual property (IP system set up to privatize it. The tactics used to design BabyLegs as a feminine, silly, doll-tool to discredit the device in the eyes of an IP system that valued traditional gender roles lead to the eventual success of keeping the device open source. Yet, those same tactics also reinforced and reproduced the structures of power and essentialism they were designed to resist. I characterize this technological ambivalence as compromise, and argue that all agency exercised within asymmetrical power relations is compromised. This is not to say resistance is futile, but that agency is never pure, and this recognition lets us be more intentional in how we might compromise as practitioners of diverse scientific knowledges.

  16. Comparison of protease production from newly isolated bacterial ...

    African Journals Online (AJOL)

    Nasir

    2016-10-12

    Oct 12, 2016 ... Protease has gained a very important position in many industries such as food, pharmaceutical, chemical and leather industries. In this research, protease was obtained from bacteria. The bacterial strain was obtained from soil which was collected from different areas of Lahore, Pakistan. Fermentation ...

  17. Oxidative Stress: Promoter of Allergic Sensitization to Protease Allergens?

    NARCIS (Netherlands)

    van Rijt, Leonie S.; Utsch, Lara; Lutter, René; van Ree, Ronald

    2017-01-01

    Allergies arise from aberrant T helper type 2 responses to allergens. Several respiratory allergens possess proteolytic activity, which has been recognized to act as an adjuvant for the development of a Th2 response. Allergen source-derived proteases can activate the protease-activated receptor-2,

  18. Alkaline protease production on date waste by an alkalophilic ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

    May 16, 2008 ... After 72 h incubation in a shaker incubator ... different incubation times (0 to 72 h) were investigated. Alkaline .... of alkaline protease (75%) and 24% of total protein is precipitated. ... starches and wheat flour as carbon source on protease production .... JP 395, method of making and detergent composition.

  19. Extracellular protease produced by Bacillus subtilis isolated from ...

    African Journals Online (AJOL)

    In a study to evaluate the microbiological safety of some paracetamol oral solutions sold in some Nigerian drug stores, 40.0% of the samples examined was contaminated with protease-producing Bacillus subtilis. The production of extracellular protease was induced by casein in the minimal medium and was found to be the ...

  20. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

    Aug 28, 2012 ... production proved high protease production than the other tested ... Crude alkaline protease was most active at 55°C, pH 9 with casein as ... 13416 Afr. J. Biotechnol. ... The Gram-positive, aerobic, rod-shaped endospore-.

  1. Model building of a thermolysin-like protease by mutagenesis

    NARCIS (Netherlands)

    Frigerio, F; Margarit, [No Value; Nogarotto, R; Grandi, G; Vriend, G; Hardy, F; Veltman, OR; Venema, G; Eijsink, VGH

    The present study concerns the use of site-directed mutagenesis experiments to optimize a three-dimensional model of the neutral protease of Bacillus subtilis (NP-sub), An initial model of NP-sub was constructed using the crystal structures of the homologous neutral proteases of Bacillus

  2. Cold denaturation of the HIV-1 protease monomer

    DEFF Research Database (Denmark)

    Rösner, Heike Ilona; Caldarini, Martina; Prestel, Andreas

    2017-01-01

    The HIV-1-protease is a complex protein which in its active form adopts a homodimer dominated by -sheet structures. We have discovered a cold-denatured state of the monomeric subunit of HIV-1-protease which is populated above 0ºC and therefore directly accessible to various spectroscopic approac...

  3. Oxidant and solvent stable alkaline protease from Aspergillus flavus ...

    African Journals Online (AJOL)

    The increase in agricultural practices has necessitated the judicious use of agricultural wastes into value added products. In this study, an extracellular, organic solvent and oxidant stable, serine protease was produced by Aspergillus flavus MTCC 9952 under solid state fermentation. Maximum protease yield was obtained ...

  4. Some physicochemical properties of acid protease produced during ...

    African Journals Online (AJOL)

    The growth of Aspergillus niger (NRRL 1785) was investigated and monitored over a five-day fermentation period. Acid protease synthesis by this fungus was also investigated during the period. The effect of growth of Aspergillus niger on acid protease synthesis was determined. Some of the physicochemical properties of ...

  5. Improvement of acid protease production by a mixed culture of ...

    African Journals Online (AJOL)

    The synthesis of acid protease by Aspergillus oryzae AS3042 was enhanced significantly with the mixed culture of Aspergillus niger SL-09 using solid-state fermentation technique. The influence of carbon sources, nitrogen sources and the addition of phytic acid on acid protease production were investigated. The enzyme ...

  6. Partial purification and characterization of alkaline proteases from ...

    African Journals Online (AJOL)

    Alkaline proteases from the digestive tract of anchovy were partially purified by ammonium sulfate fractionation, dialysis and Sephadex G-75 gel filtration. The purification fold and yield were 6.23 and 4.49%, respectively. The optimum activities of partially purified alkaline proteases were observed at 60°C and at pH 11.0.

  7. High-level expression of alkaline protease using recombinant ...

    African Journals Online (AJOL)

    AJL

    2012-02-16

    Feb 16, 2012 ... compared with that of wild-type B. licheniformis CICIM B5102. Key word: Alkaline protease, Bacillus amyloliquefaciens, Bacillus licheniformis. INTRODUCTION. Proteases are one of the most important industrial enzyme groups, accounting for approximately 60% of the total enzyme sales (Beg et al., 2003).

  8. Isolation of protease producing novel Bacillus cereus and detection ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... The highest protease activity was determined at 30°C temperature and 6.4 pH conditions and after the 18th hour, it decreased evidently. Key words: Protease, production, optimization, Bacillus sp. INTRODUCTION. Enzymes have been produced in large industrial scale for several decades (Falch, 1991).

  9. Production of alkaline proteases by alkalophilic Bacillus subtilis ...

    African Journals Online (AJOL)

    Among various nitrogen sources, yeast extract was found to be the best inducer of alkaline protease. Among metal salts, KNO3 and NH4Cl were found to increase protease production. The maximum enzyme production (3600 U/ml) was observed with pomegranate peels of fermentation medium in the presence of yeast ...

  10. Assessment of microbial activity and biomass in different soils exposed to nicosulfuron

    Directory of Open Access Journals (Sweden)

    Ljiljana Šantrić

    2014-09-01

    Full Text Available The effects of the herbicide nicosulfuron on the abundance of cellulolytic and proteolytic microorganisms, activity of β-glucosidase and protease enzymes, and microbial phosphorus biomass were examined. A laboratory bioassay was set up on two types of agricultural soils differing in physicochemical properties. The following concentrations were tested: 0.3, 0.6, 3.0 and 30.0 mg a.i./kg of soil. Samples were collected 3, 7, 14, 30 and 45 days after treatment with nicosulfuron. The results showed that nicosulfuron significantly reduced the abundance of cellulolytic microorganisms in both soils, as well as microbial biomass phosphorus in sandy loam soil. The herbicide was found to stimulate β-glucosidase and protease activity in both types of soil and microbial biomass phosphorus in loamy soil. Proteolytic microorganisms remained unaffected by nicosulfuron.

  11. Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases.

    Science.gov (United States)

    Panigrahi, Rashmi; Lemieux, M Joanne

    2015-01-01

    Intramembrane proteases are membrane embedded enzymes that cleave transmembrane substrates. This interesting class of enzyme and its water mediated substrate cleavage mechanism occurring within the hydrophobic lipid bilayer has drawn the attention of researchers. Rhomboids are a family of ubiquitous serine intramembrane proteases. Bacterial forms of rhomboid proteases are mainly composed of six transmembrane helices that are preceded by a soluble N-terminal domain. Several crystal structures of the membrane domain of the E. coli rhomboid protease ecGlpG have been solved. Independently, the ecGlpG N-terminal cytoplasmic domain structure was solved using both NMR and protein crystallography. Despite these structures, we still do not know the structure of the full-length protein, nor do we know the functional role of these domains in the cell. This chapter will review the structural and functional roles of the different domains associated with prokaryotic rhomboid proteases. Lastly, we will address questions remaining in the field.

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

    Science.gov (United States)

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

    2009-01-01

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

  13. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Directory of Open Access Journals (Sweden)

    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  14. Microbial safety of foods

    International Nuclear Information System (INIS)

    Bandekar, J.R.

    2013-01-01

    Despite advances in hygiene, consumer knowledge and food treatment and processing, food-borne diseases have become one of the most widespread public health problems in the world to-day. About two thirds of all outbreaks are traced to microbial contaminated food - one of the most hazardous being Clostridium botulinum, E. coli 0157: H7 and Salmonella. The pathogens can be introduced in the food products anywhere in the food chain and hence it is of prime important to have microbial vigilance in the entire food chain. WHO estimates that food-borne and water-borne diarrhoeal diseases taken together kill about 2.2 million people annually. The infants, children, elderly and immune-compromised people are particularly susceptible to food-borne diseases. Unsafe food causes many acute and life-long diseases, ranging from diarrhoeal diseases to various forms of cancer. A number of factors such as emergence of new food-borne pathogens, development of drug resistance in the pathogens, changing life style, global trade of food etc. are responsible for the continued persistence of food-borne diseases. Due to consumer demand, a number of Ready-To-Eat (RTE) minimally processed foods are increasingly marketed. However, there is increased risk of food-borne diseases with these products. The food-borne disease outbreaks due to E. coli O157:H7, Listeria monocytogenes, Salmonella and Campylobacter are responsible for recall of many foods resulting in heavy losses to food industry. The development of multi drug resistant pathogens due to indiscriminate use of antibiotics is also a major problem. Food Technology Division of Bhabha Atomic Research Centre has been working on food-borne bacterial pathogens particularly Salmonella, Campylobacter, Vibrio and Aeromonas species, their prevalence in export quality seafood as well in foods sold in retail market such as poultry, fish, sprouts and salads. These pathogens from Indian foods have been characterized for the presence of virulence genes

  15. Reduction of protease activity in milk by continuous flow high-intensity pulsed electric field treatments.

    Science.gov (United States)

    Bendicho, S; Barbosa-Cánovas, G V; Martín, O

    2003-03-01

    High-intensity pulsed electric field (HIPEF) is a non-thermal food processing technology that is currently being investigated to inactivate microorganisms and certain enzymes, involving a limited increase of food temperature. Promising results have been obtained on the inactivation of microbial enzymes in milk when suspended in simulated milk ultrafiltrate. The aim of this study was to evaluate the effectiveness of continuous HIPEF equipment on inactivating a protease from Bacillus subtilis inoculated in milk. Samples were subjected to HIPEF treatments of up to 866 micros of squared wave pulses at field strengths from 19.7 to 35.5 kV/cm, using a treatment chamber that consisted of eight colinear chambers connected in series. Moreover, the effects of different parameters such as pulse width (4 and 7 micros), pulse repetition rates (67, 89, and 111 Hz), and milk composition (skim and whole milk) were tested. Protease activity decreased with increased treatment time or field strength and pulse repetition rate. Regarding pulse width, no differences were observed between 4 and 7 micros pulses when total treatment time was considered. On the other hand, it was observed that milk composition affected the results since higher inactivation levels were reached in skim than in whole milk. The maximum inactivation (81%) was attained in skim milk after an 866-micros treatment at 35.5 kV/cm and 111 Hz.

  16. Highly potent fibrinolytic serine protease from Streptomyces.

    Science.gov (United States)

    Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

    2011-01-05

    We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. Copyright © 2010 Elsevier Inc. All rights reserved.

  17. Characterization of a secreted Chlamydia protease

    DEFF Research Database (Denmark)

    Shaw, A.C.; Vandahl, B.B.; Larsen, M.R.

    2002-01-01

    Chlamydiae are obligate intracellular bacteria that are important human pathogens. The Chlamydia genomes contain orthologues to secretion apparatus proteins from other intracellular bacteria, but only a few secreted proteins have been identified. Most likely, effector proteins are secreted in order...... to promote infection. Effector proteins cannot be identified by motif or similarity searches. As a new strategy for identification of secreted proteins we have compared 2D-PAGE profiles of [35S]-labelled Chlamydia proteins from whole lysates of infected cells to 2D-PAGE profiles of proteins from purified...... Chlamydia. Several secretion candidates from Chlamydia trachomatis D and Chlamydia pneumoniae were detected by this method. Two protein spots were identified among the candidates. These represent fragments of the 'chlamydial protease- or proteasome-like activity factor' (CPAF) and were clearly present in 2D...

  18. Diagnosis of hemodynamic compromise in patients with chronic cerebral ischemia

    International Nuclear Information System (INIS)

    Kuroda, Satoshi; Sakuragi, Mitsugi; Motomiya, Mineo; Nakagawa, Tango; Mitsumori, Kenji; Tsuru, Mitsuo; Takigawa, Shugo; Kamiyama, Hiroyasu; Abe, Hiroshi.

    1990-01-01

    To evaluate the efficacy of tests for selecting patients with hemodynamic compromise, measurement of cerebral blood volume (CBV) with 99m Tc-RBC single photon emission computed tomography (SPECT) was performed in thirteen patients with occlusive cerebrovascular disease, and was compared with results obtained by 133 Xe SPECT and acetazolamide (Diamox) test. All patients in our study suffered TIA, RIND, or minor completed stroke. Cerebral angiography demonstrated severe stenosis or occlusion in the ipsilateral internal carotid artery or middle cerebral artery, although plain CT scan or MRI revealed no or, if any, only localized infarcted lesions. Regional cerebral blood volume (rCBV) was measured with 99m Tc-RBC SPECT and regional cerebral blood flow (rCBF) was measured with 133 Xe SPECT before and after intravenous injection of 10 - 12 mg/kg acetazolamide (Diamox). Our results suggest that the ipsilateral rCBV/rCBF (mean transit time) is a more sensitive index of the cerebral perfusion reserve than the use of only rCBV or rCBF of the ipsilateral hemisphere. Also, the ipsilateral rCBV/rCBF is significantly correlated (r= -0.72) with the Diamox reactivity of rCBF, which is considered to represent the cerebral vasodilatory capacity in patients with chronic cerebral ischemia. Postoperative SPECT study revealed remarkable improvement of ipsilateral rCBV/rCBF and Diamox reactivity in four patients who underwent EC/IC bypass surgery to improve the hemodynamic compromise. In conclusion, our results suggest that the measurement of rCBV/rCBF with 133 Xe SPECT and 99m Tc-RBC SPECT is useful for detecting the hemodynamic compromise in patients with occlusive cerebrovascular disease. (author)

  19. Osseous pseudo-myelomatose compromise, in leukemia chronic lymphoid

    International Nuclear Information System (INIS)

    Martinez Betancur, Octavio; Lopez de Goenaga, Maria Ines

    2000-01-01

    It was described a case of chronic lymphocytic leukemia in a 75 year old man, with pseudomyelomatosis osteolytic lesions in the skull, excluding other potential causes of osteolytic lesions in the clinical context of malignant lymphoproliferative neoplasm. The real frequency of osseous compromise in chronic lymphocytic leukemia is 10%. Lesions are defined as generalized osteoporosis and osteolysis with lacunar aspect, similar to myeloma lesions. Because histopathology in lymphoproliferative neoplasms may be similar, it might be difficult to diagnose chronic lymphocytic leukemia certainly, if the clinical manifestations are not considered. Differential diagnosis with other lymphoproliferative neoplasm is based basically in absolute lymphocytosis greater than 10 X 109/L, with lymphocytes with mature appearance

  20. The Swiss approach to finding compromises in nuclear waste governance

    Energy Technology Data Exchange (ETDEWEB)

    Kuppler, Sophie; Grunwald, Armin [Karlsruhe Institute of Technology (Germany). Inst. for Technology Assessment and Systems Analysis

    2015-07-01

    In Switzerland, a new site selection procedure is being implemented since 2008. This procedure, which is laid down in a 'sectoral plan', shows strong elements of public participation and transparency and can be considered a step away from the classical 'decide-announce-defend' approach in decision-making. This procedure tends towards a more governance-oriented approach based on ideas of 'civility' of decision-making. Despite this renewal, the Swiss case clearly shows that any kind of selection process has to be considered as a 'working compromise', which needs to be adapted when new challenges emerge.

  1. The Swiss approach to finding compromises in nuclear waste governance

    International Nuclear Information System (INIS)

    Kuppler, Sophie; Grunwald, Armin

    2015-01-01

    In Switzerland, a new site selection procedure is being implemented since 2008. This procedure, which is laid down in a 'sectoral plan', shows strong elements of public participation and transparency and can be considered a step away from the classical 'decide-announce-defend' approach in decision-making. This procedure tends towards a more governance-oriented approach based on ideas of 'civility' of decision-making. Despite this renewal, the Swiss case clearly shows that any kind of selection process has to be considered as a 'working compromise', which needs to be adapted when new challenges emerge.

  2. Structure of HIV-1 protease determined by neutron crystallography

    International Nuclear Information System (INIS)

    Adachi, Motoyasu; Kuroki, Ryota

    2009-01-01

    HIV-1 protease is an aspartic protease, and plays an essential role in replication of HIV. To develop HIV-1 protease inhibitors through structure-based drug design, it is necessary to understand the catalytic mechanism and inhibitor recognition of HIV-1 protease. We have determined the crystal structure of HIV-1 protease in complex with KNI-272 to 1.9 A resolution by neutron crystallography in combination with 1.4 A resolution X-ray diffraction data. The results show that the carbonyl group of hydroxymethylcarbonyl (HMC) in KNI-272 forms a hydrogen bonding interaction with protonated Asp 25 and the hydrogen atom from the hydroxyl group of HMC forms a hydrogen bonding interaction with the deprotonated Asp125. This is the first neutron report for HIV-1/inhibitor complex and shows directly the locations of key hydrogen atoms in catalysis and in the binding of a transition-state analog. The results confirm key aspect of the presumed catalytic mechanism of HIV-1 protease and will aid in the further development of protease inhibitors. (author)

  3. Bacillus amyloliquefaciens SUBSP. plantarum PROBIOTIC STRAINS AS PROTEASE PRODUCERS

    Directory of Open Access Journals (Sweden)

    E. V. Маtseliukh

    2015-04-01

    Full Text Available Proteases from probiotic strains of the genus Bacillus, just like the antibiotics, bacteriocins and other hydrolytic enzymes, are one of the main factors that determine their biological activity. The aim of this work was to study the synthesis and biochemical properties of proteases from two strains Bacillus amyloliquefaciens subsp. plantarum UCM B-5139 and UCM B-5140 that included in the probiotic Endosporin. The cultivation of strains was carried out in flasks under rotating for two days. The influence of physico-chemical parameters of the reaction medium on proteolytic activity was studied on partially purified protease preparations. Lytic activity was determined by turbidimetric method. On the second day of cultivation B. amyloliquefaciens subsp. plantarum UCM В-5139 and UCM В-5140 synthesized the metal-dependent peptidase and serine protease, respectively. The optimum conditions of their action were the following: temperature 37–40 °C and pH 6.5–7.0. Isolated proteases are able to lyse the living cells of Staphylococcus aureus and Candida albicans. Thus we demonstrated that B. amyloliquefaciens subsp. plantarum UCM B-5140 and UCM B-5139, included in the probiotic veterinary preparation Endosporin, produced proteolytic enzymes that hydrolyze the native insoluble proteins (elastin, fibrin and collagen. These enzymes belong to the group of neutral metal-dependent and serine proteases. They are active under physiological conditions against gram-positive bacteria and yeasts. The application of these proteases in biotechnology is considered.

  4. Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

    Science.gov (United States)

    Wilkesman, Jeff; Padrón, María Fernanda; Kurz, Liliana; Rémignon, Hervé

    2017-01-01

    Protease activity present in liver cells with steatosis can be electrophoretically characterized. Zymographic techniques allow semi-quantitative results, successfully detecting cathepsin and metalloprotease activity using polyacrylamide gels copolymerized with gelatin and quantified by densitometry. By using specific inhibitors, the identity of the proteases can be confirmed. 2D zymography allows the determination of both M r. and pI of the metalloprotease and cathepsin activity present in the homogenates. The analysis of liver proteases activities in force fed ducks may elucidate the mechanisms behind steatosis development.

  5. Hyper production of alkaline protease by mutagenized bacillus subtilis

    International Nuclear Information System (INIS)

    Qureshi, A.M.; Tanseem, F.

    2010-01-01

    The purpose of this work was to augment the alkaline protease production from Bacillus subtilis by using chemical mutagen (MMS) and UV mutagenesis. A number of mutants were isolated which produce high levels of extra cellular proteases. Analysis of culture supernatants of these mutants had shown that the total amounts of proteolysis activity were increased from 1 to 2 fold over the wild strain. Clones showing promote response were further characterized by analyzing different parameters; like of Temperature, pH substrate concentration and incubation period, to study the activity of protease enzyme. (author)

  6. Optimizing PHB and Protease Production by Box Behnken Design

    Directory of Open Access Journals (Sweden)

    Amro Abd al fattah Amara

    2013-04-01

    Full Text Available Mixed culture is more suitable to adapt more flexible fermentation process and produce different product simultaneously. In this study a mixed Bacillus culture was investigated for their ability to produce the bioplastic "Polyhydroxybutyrate" and both of the mesophilic and the thermophilic proteases in one flask. Box-Behnken experimental design was used. The produced amount of PHB has been increased significantly. Meanwhile there is a competition between PHB and proteases. The maximum produced amount of PHB using Box-Behnken design was 2.82 g/l/48 h with protease activity equal to 41.9 Units/ml/48 h for thermophilic proteases and 99.65 Units/ml/48 h for mesophilic proteases. Excel solver was used for extra-optimization for the optimum conditions obtained from Box-Behnken experiments and its model. The maximum PHB obtained after using Excel solver was 2.88 g/l/48 h. The maximum mesophilic and thermophilic activities obtained at the same PHB production conditions were 175.68 and 243.38 Units/ml respectively. The model accuracy as obtained from Excel solver was 118.8%, which prove the power of the experimental design in optimizing such complicated process. The strategies used in this study are recommended for the production of PHB and different proteases simultaneously using Bacillus mixed culture. ABSTRAK: Kultur campuran adalah lebih sesuai bagi proses penapaian yang fleksibel dan ia boleh menghasilkan produk yang berbeza secara serentak. Dalam kajian ini keupayaan  menghasilkan "Polyhydroxybutyrate" bioplastik serta mesofilik dan termofilik protease dalam satu flask oleh  kultur Bacillus campuran telah disiasat. Eksperimen rekabentuk Box-Behnken telah digunakan. Jumlah PHB yang dikeluarkan meningkat dengan ketara dan terdapat persaingan antara PHB dan protease. Jumlah keluaran PHB maksima menggunakan rekabentuk Box-Behnken adalah 2.82 g/l/48 jam dengan aktiviti protease sama dengan 41.9 Unit/ml/48 jam untuk protease termofilik dan 99.65 Unit

  7. Protease-associated cellular networks in malaria parasite Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    Lilburn Timothy G

    2011-12-01

    Full Text Available Abstract Background Malaria continues to be one of the most severe global infectious diseases, responsible for 1-2 million deaths yearly. The rapid evolution and spread of drug resistance in parasites has led to an urgent need for the development of novel antimalarial targets. Proteases are a group of enzymes that play essential roles in parasite growth and invasion. The possibility of designing specific inhibitors for proteases makes them promising drug targets. Previously, combining a comparative genomics approach and a machine learning approach, we identified the complement of proteases (degradome in the malaria parasite Plasmodium falciparum and its sibling species 123, providing a catalog of targets for functional characterization and rational inhibitor design. Network analysis represents another route to revealing the role of proteins in the biology of parasites and we use this approach here to expand our understanding of the systems involving the proteases of P. falciparum. Results We investigated the roles of proteases in the parasite life cycle by constructing a network using protein-protein association data from the STRING database 4, and analyzing these data, in conjunction with the data from protein-protein interaction assays using the yeast 2-hybrid (Y2H system 5, blood stage microarray experiments 678, proteomics 9101112, literature text mining, and sequence homology analysis. Seventy-seven (77 out of 124 predicted proteases were associated with at least one other protein, constituting 2,431 protein-protein interactions (PPIs. These proteases appear to play diverse roles in metabolism, cell cycle regulation, invasion and infection. Their degrees of connectivity (i.e., connections to other proteins, range from one to 143. The largest protease-associated sub-network is the ubiquitin-proteasome system which is crucial for protein recycling and stress response. Proteases are also implicated in heat shock response, signal peptide

  8. Gamma processing of Arabic bread for immune system-compromised cancer patients

    International Nuclear Information System (INIS)

    Grecz, N.; Brannon, R.; Jaw, R.; Al-Harithy, R.; Hahn, E.W.

    1985-01-01

    Arabic bread prepared from local Saudi flour contained a total of up to 105 4 organisms per g. Most of these were bacterial spores that survived the baking process (1.3 x 10 2 to 3.5 x 10 3 ) and a small number of yeasts and molds (10 to 40 cells per g). The organisms in Arabic bread appear to be harmless to healthy individuals. However, for immune system-compromised cancer patients and bone marrow transplant recipients, it is prudent to irradiate the bread to reduce microbial contamination. The decimal reduction doses (10% survival) for the most radiation-resistant organisms (spore formers) in bread were 0.11 to 0.15 Mrad. Accordingly, 0.6 Mrad was sufficient to reduce the number of spores in Arabic bread by a factor of 10,000, i.e., to <1/g. This treatment constitutes radiation pasteurization (radicidation), and to this extent, provides a margin of microbiological safety. Sensory evaluation by the nine-point hedonic scale showed no detectable loss of organoleptic quality of bread up to 0.6 Mrad, while irradiation to 2.5 Mrad induced unacceptable organoleptic changes

  9. Protozoan predation in soil slurries compromises determination of contaminant mineralization potential

    International Nuclear Information System (INIS)

    Badawi, Nora; Johnsen, Anders R.; Brandt, Kristian K.; Sørensen, Jan; Aamand, Jens

    2012-01-01

    Soil suspensions (slurries) are commonly used to estimate the potential of soil microbial communities to mineralize organic contaminants. The preparation of soil slurries disrupts soil structure, however, potentially affecting both the bacterial populations and their protozoan predators. We studied the importance of this “slurry effect” on mineralization of the herbicide 2-methyl-4-chlorophenoxyacetic acid (MCPA, 14 C-labelled), focussing on the effects of protozoan predation. Mineralization of MCPA was studied in “intact” soil and soil slurries differing in soil:water ratio, both in the presence and absence of the protozoan activity inhibitor cycloheximide. Protozoan predation inhibited mineralization in dense slurry of subsoil (soil:water ratio 1:3), but only in the most dilute slurry of topsoil (soil:water ratio 1:100). Our results demonstrate that protozoan predation in soil slurries may compromise quantification of contaminant mineralization potential, especially when the initial density of degrader bacteria is low and their growth is controlled by predation during the incubation period. - Highlights: ► We studied the protozoan impact on MCPA mineralization in soil slurries. ► Cycloheximide was used as protozoan inhibitor. ► Protozoa inhibited MCPA mineralization in dilute topsoil slurry and subsoil slurry. ► Mineralization potentials may be underestimated when using soil slurries. - Protozoan predation may strongly bias the quantification of mineralization potential when performed in soil slurries, especially when the initial density of degrader bacteria is low such as in subsoil or very dilute topsoil slurries.

  10. A novel lung slice system with compromised antioxidant defenses

    Energy Technology Data Exchange (ETDEWEB)

    Hardwick, S.J.; Adam, A.; Cohen, G.M. (Univ. of London (England)); Smith, L.L. (Imperial Chemical Industries PLC, Cheshire (England))

    1990-04-01

    In order to facilitate the study of oxidative stress in lung tissue, rat lung slices with impaired antioxidant defenses were prepared and used. Incubation of lung slices with the antineoplastic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 {mu}M) in an amino acid-rich medium for 45 min produced a near-maximal (approximately 85%), irreversible inhibition of glutathione reductase, accompanied by only a modest (approximately 15%) decrease in pulmonary nonprotein sulfhydryls (NPSH) and no alteration in intracellular ATP, NADP{sup +}, and NADPH levels. The amounts of NADP(H), ATP, and NPSH were stable over a 4-hr incubation period following the removal from BCNU. The viability of the system was further evaluated by measuring the rate of evolution of {sup 14}CO{sub 2} from D-({sup 14}C(U))-glucose. The rates of evolution were almost identical in the compromised system when compared with control slices over a 4-hr time period. By using slices with compromised oxidative defenses, preliminary results have been obtained with paraquat, nitrofurantoin, and 2,3-dimethoxy-1,4-naphthoquinone.

  11. Anthropogenic noise compromises antipredator behaviour in European eels.

    Science.gov (United States)

    Simpson, Stephen D; Purser, Julia; Radford, Andrew N

    2015-02-01

    Increases in noise-generating human activities since the Industrial Revolution have changed the acoustic landscape of many terrestrial and aquatic ecosystems. Anthropogenic noise is now recognized as a major pollutant of international concern, and recent studies have demonstrated impacts on, for instance, hearing thresholds, communication, movement and foraging in a range of species. However, consequences for survival and reproductive success are difficult to ascertain. Using a series of laboratory-based experiments and an open-water test with the same methodology, we show that acoustic disturbance can compromise antipredator behaviour--which directly affects survival likelihood--and explore potential underlying mechanisms. Juvenile European eels (Anguilla anguilla) exposed to additional noise (playback of recordings of ships passing through harbours), rather than control conditions (playback of recordings from the same harbours without ships), performed less well in two simulated predation paradigms. Eels were 50% less likely and 25% slower to startle to an 'ambush predator' and were caught more than twice as quickly by a 'pursuit predator'. Furthermore, eels experiencing additional noise had diminished spatial performance and elevated ventilation and metabolic rates (indicators of stress) compared with control individuals. Our results suggest that acoustic disturbance could have important physiological and behavioural impacts on animals, compromising life-or-death responses. © 2014 John Wiley & Sons Ltd.

  12. A novel lung slice system with compromised antioxidant defenses

    International Nuclear Information System (INIS)

    Hardwick, S.J.; Adam, A.; Cohen, G.M.; Smith, L.L.

    1990-01-01

    In order to facilitate the study of oxidative stress in lung tissue, rat lung slices with impaired antioxidant defenses were prepared and used. Incubation of lung slices with the antineoplastic agent 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) (100 μM) in an amino acid-rich medium for 45 min produced a near-maximal (approximately 85%), irreversible inhibition of glutathione reductase, accompanied by only a modest (approximately 15%) decrease in pulmonary nonprotein sulfhydryls (NPSH) and no alteration in intracellular ATP, NADP + , and NADPH levels. The amounts of NADP(H), ATP, and NPSH were stable over a 4-hr incubation period following the removal from BCNU. The viability of the system was further evaluated by measuring the rate of evolution of 14 CO 2 from D-[ 14 C(U)]-glucose. The rates of evolution were almost identical in the compromised system when compared with control slices over a 4-hr time period. By using slices with compromised oxidative defenses, preliminary results have been obtained with paraquat, nitrofurantoin, and 2,3-dimethoxy-1,4-naphthoquinone

  13. Differential diagnosis of the signal-compromised lunate in MRI

    International Nuclear Information System (INIS)

    Schmitt, R.; Christopoulos, G.; Coblenz, G.; Froehner, S.; Brunner, H.; Kalb, K.; Krimmer, H.; Lanz, U.

    2005-01-01

    Purpose: To define both the underlying pathology and diagnostic criteria in lunates presenting with conspicuous signal pattern in MRI. Materials and Methods: The retrospective evaluation of 2940 MRI examinations revealed 203 patients with signal alterations of the lunate. All MRI examinations were performed on 1.5-Tesla platforms using dedicated surface coils and an intravenous contrast agent. To establish a definitive diagnosis, a total of 252 MRI examinations (49 follow-ups), 22 CT examinations and 4 arthroscopic studies were obtained in addition to the obligatory conventional radiographs. Results: Incorporating all clinical data, radiographs and MRI examinations succeeded in assigning a diagnosis in 136 signal-compromised lunates (67.0%), whereas additional diagnostic procedures or follow-up examinations were required for the definitive diagnosis in 57 cases (33.0%). The most frequent entities were 51 cases of Kienboeck's disease (25.1%), 47 cases of ulnolunate-(triquetral) impaction syndromes (23.2%) and 44 cases of intra-osseous ganglion cysts (21.7%). Other pathologies included 23 degenerative, 19 traumatic and 10 inflammatory changes as well as 9 congenital conditions. For MRI assessment of the altered lunate, the most important parameters were location and morphology as well as involvement of the articular and osseous structures of the carpus. Conclusion: The lunate may be affected by different pathological states of the wrist. In total, only one quarter of the signal-compromised lunate represented Kienboeck's disease. (orig.)

  14. Parental care compromises feeding in the pumpkinseed ( Lepomis gibbosus)

    Science.gov (United States)

    Zięba, G.; Dukowska, M.; Przybylski, M.; Fox, M. G.; Smith, C.

    2018-04-01

    Providing parental care is potentially costly. Costs can arise through elevated energy expenditure or from an increased risk of mortality. A cost of parental care can also occur because a parent is compromised in their ability to forage. We used pumpkinseed Lepomis gibbosus, a fish with an alternative male mating strategy, to test whether parental males differed in their feeding in comparison with females and cuckolder males. To address this question, we examined the stomach contents of female, cuckolder male, and parental male pumpkinseed during the breeding season over an entire diel cycle. We showed that parental males had a lower total weight of food in their stomachs in comparison with females, while cuckolder males did not. Parental males also had a lower weight and number of chironomids in their stomachs. The temporal pattern of feeding of parental males diverged from that of females, and they had a lower probability of pupal chironomids in their stomachs, which implies spatial segregation in foraging. Parental males had a greater probability of conspecific eggs in their stomachs than females, while the probability of egg cannibalism did not differ between cuckolder males and females. Overall, these finding meet predictions in accordance with an assumption that parental care and territoriality can compromise feeding.

  15. Genome-wide identification and structure-function studies of proteases and protease inhibitors in Cicer arietinum (chickpea).

    Science.gov (United States)

    Sharma, Ranu; Suresh, C G

    2015-01-01

    Proteases are a family of enzymes present in almost all living organisms. In plants they are involved in many biological processes requiring stress response in situations such as water deficiency, pathogen attack, maintaining protein content of the cell, programmed cell death, senescence, reproduction and many more. Similarly, protease inhibitors (PIs) are involved in various important functions like suppression of invasion by pathogenic nematodes, inhibition of spores-germination and mycelium growth of Alternaria alternata and response to wounding and fungal attack. As much as we know, no genome-wide study of proteases together with proteinaceous PIs is reported in any of the sequenced genomes till now. Phylogenetic studies and domain analysis of proteases were carried out to understand the molecular evolution as well as gene and protein features. Structural analysis was carried out to explore the binding mode and affinity of PIs for cognate proteases and prolyl oligopeptidase protease with inhibitor ligand. In the study reported here, a significant number of proteases and PIs were identified in chickpea genome. The gene expression profiles of proteases and PIs in five different plant tissues revealed a differential expression pattern in more than one plant tissue. Molecular dynamics studies revealed the formation of stable complex owing to increased number of protein-ligand and inter and intramolecular protein-protein hydrogen bonds. The genome-wide identification, characterization, evolutionary understanding, gene expression, and structural analysis of proteases and PIs provide a framework for future analysis when defining their roles in stress response and developing a more stress tolerant variety of chickpea. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Microbial micropatches within microbial hotspots

    Science.gov (United States)

    Smith, Renee J.; Tobe, Shanan S.; Paterson, James S.; Seymour, Justin R.; Oliver, Rod L.; Mitchell, James G.

    2018-01-01

    The spatial distributions of organism abundance and diversity are often heterogeneous. This includes the sub-centimetre distributions of microbes, which have ‘hotspots’ of high abundance, and ‘coldspots’ of low abundance. Previously we showed that 300 μl abundance hotspots, coldspots and background regions were distinct at all taxonomic levels. Here we build on these results by showing taxonomic micropatches within these 300 μl microscale hotspots, coldspots and background regions at the 1 μl scale. This heterogeneity among 1 μl subsamples was driven by heightened abundance of specific genera. The micropatches were most pronounced within hotspots. Micropatches were dominated by Pseudomonas, Bacteroides, Parasporobacterium and Lachnospiraceae incertae sedis, with Pseudomonas and Bacteroides being responsible for a shift in the most dominant genera in individual hotspot subsamples, representing up to 80.6% and 47.3% average abundance, respectively. The presence of these micropatches implies the ability these groups have to create, establish themselves in, or exploit heterogeneous microenvironments. These genera are often particle-associated, from which we infer that these micropatches are evidence for sub-millimetre aggregates and the aquatic polymer matrix. These findings support the emerging paradigm that the microscale distributions of planktonic microbes are numerically and taxonomically heterogeneous at scales of millimetres and less. We show that microscale microbial hotspots have internal structure within which specific local nutrient exchanges and cellular interactions might occur. PMID:29787564

  17. Isolasi, Seleksi Dan Opttmasi Produksi Protease Daribeberapaisolat Bakteri*(isolation, Selection and Optimalization of Protease Production of Some Bacterial Isolates)

    OpenAIRE

    Naiola, Elidar; Widhyastuti, Nunuk

    2002-01-01

    Thirty-seven out of sixty-one bacterial isolates from various sources of samples were screened for protease production. The isolate of ISO PL3 could produce the highest enzyme activity, and it was used as a standard bacterial strain in this observation. For any reason,we implemented ISO PL2 to study the optimum condition for producing bacterial protease. Result shows that the maximum protease activity was obtained in a medium containing 100 gram of rice brand in a liter tofu liquid waste. The...

  18. Cloning and characterization of a novel cysteine protease gene ...

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

    Cysteine proteases can be found in the animal and plant kingdoms as well as in some viruses and bacteria. They have been implemented in many ..... in developing resistance against pathogens and insects in other crops. Acknowledgments.

  19. Purification and characterization of a protease from Thermophilic ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-10-19

    PAGE ... applications has been well recognized and it was ... One particular interest is the production of alkaline protease from bacillus for applications in detergent industry. (Ferrero et al., 1996; Manachini and Fortina, 1998;.

  20. Immune pressure analysis of protease and reverse transcriptase ...

    African Journals Online (AJOL)

    /dn) were analyzed for 33 HIV-1 subtype C protease (PR) and reverse transcriptase (RT) nucleotide sequences each from antiretroviral naïve South African chronically infected individuals. The ds/dn ratios were calculated using the ...

  1. A Protease Isolated from the Latex of Plumeria rubra Linn ...

    African Journals Online (AJOL)

    Erah

    purified protease was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-. PAGE). ... by ammonium sulphate (40 - 60% w/v). The solution was kept .... chloride (88.1 %), silver nitrate (92.9 %), mercuric chloride ...

  2. 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.

  3. Effect of Gastrointestinal Protease Digestion on Bioactivity of Marine Peptides

    DEFF Research Database (Denmark)

    Jensen, Ida-Johanne; Andersen, Lisa Lystbæk; Ossum, Carlo Gunnar

    2014-01-01

    executed without concerning subsequent digestion after intake and the aim of this work was hence to investigate how the in vitro antioxidative, antihypertensive and caspase activating activities of peptides are affected by digestion with gastrointestinal (GI) proteases. Five different fish protein...... hydrolysates were chosen to study the effect of in vitro digestion on bioactivity. The protein concentration decreased in all samples during digestion and the molecular weight distribution of the peptides shifted towards lower values. Thus, in vitro digestion with GI proteases resulted in a further degradation...... of the peptides obtained by hydrolysis. The antihypertensive effect increased in all samples after digestion with GI proteases whereas the antioxidative capacity decreased. The effect on the caspase activity depended on the proteases used in the preparation of hydrolysates. In conclusion, the caspase activity...

  4. [Analysis of salivary protease spectrum in chronic periodontitis].

    Science.gov (United States)

    Qian, Li; Xuedong, Zhou; Yaping, Fan; Tengyu, Yang; Songtao, Wu; Yu, Yu; Jiao, Chen; Ping, Zhang; Yun, Feng

    2017-02-01

    This study aimed to investigate the difference in salivary protease expression in patients with chronic periodontitis and normal individuals. The stimulating saliva in patients with chronic periodontitis and normal individuals were collected. Protein chip technology was adapted to analyze salivary protease spectrum. Among the 34 proteases in the chip, disintegrin and metalloproteinase (ADAM)8, matrix metalloproteinase (MMP)-8, MMP-12, neprilysin/CD10, and uridylyl phosphate adenosine/urokinase showed a significantly increased concentration in the saliva of chronic periodontitis patients compared with those in the saliva of normal individuals (Pchronic periodontitis patients and normal individuals significantly differed. Analysis of salivary protease spectrum is a potential clinical method to examine, diagnose, and monitor chronic periodontitis.

  5. Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839

    OpenAIRE

    Sahoo, A. K.; Gaikwad, V. S.; Ranveer, R. C.; Dandge, P. B.; Waghmare, S. R.

    2016-01-01

    Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24?h of incubation with 2.5?% oil seed waste concentration. O. xanthineolytica was found to produce two proteases?P1 and P2. The proteases were purified using 60?% cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular we...

  6. Sequential Detection of Thermophilic Lipase and Protease by Zymography.

    Science.gov (United States)

    Kurz, Liliana; Hernández, Zully; Contreras, Lellys M; Wilkesman, Jeff

    2017-01-01

    Lipase and protease present in cell-free fractions of thermophilic Bacillus sp. cultures were analyzed by polyacrylamide gel (PAG) electrophoresis. After run, the gel is electrotransferred to another PAG copolymerized with glycerol tributyrate, olive oil, and gelatin. This multi-substrate gel was incubated first for lipase detection, until bands appeared, and then stained with Coomassie for protease detection. Advantages of this sequential procedure are the detection of two different enzyme activities on a single PAG, beside time and resource saving.

  7. Functional protease profiling for diagnosis of malignant disease.

    Science.gov (United States)

    Findeisen, Peter; Neumaier, Michael

    2012-01-01

    Clinical proteomic profiling by mass spectrometry (MS) aims at uncovering specific alterations within mass profiles of clinical specimens that are of diagnostic value for the detection and classification of various diseases including cancer. However, despite substantial progress in the field, the clinical proteomic profiling approaches have not matured into routine diagnostic applications so far. Their limitations are mainly related to high-abundance proteins and their complex processing by a multitude of endogenous proteases thus making rigorous standardization difficult. MS is biased towards the detection of low-molecular-weight peptides. Specifically, in serum specimens, the particular fragments of proteolytically degraded proteins are amenable to MS analysis. Proteases are known to be involved in tumour progression and tumour-specific proteases are released into the blood stream presumably as a result of invasive progression and metastasis. Thus, the determination of protease activity in clinical specimens from patients with malignant disease can offer diagnostic and also therapeutic options. The identification of specific substrates for tumour proteases in complex biological samples is challenging, but proteomic screens for proteases/substrate interactions are currently experiencing impressive progress. Such proteomic screens include peptide-based libraries, differential isotope labelling in combination with MS, quantitative degradomic analysis of proteolytically generated neo-N-termini, monitoring the degradation of exogenous reporter peptides with MS, and activity-based protein profiling. In the present article, we summarize and discuss the current status of proteomic techniques to identify tumour-specific protease-substrate interactions for functional protease profiling. Thereby, we focus on the potential diagnostic use of the respective approaches. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Sarcoidosis with Major Airway, Vascular and Nerve Compromise

    Directory of Open Access Journals (Sweden)

    Hiroshi Sekiguchi

    2013-01-01

    Full Text Available The present report describes a 60-year-old Caucasian woman who presented with progressive dyspnea, cough and wheeze. A computed tomography scan of the chest showed innumerable bilateral inflammatory pulmonary nodules with bronchovascular distribution and a mediastinal and hilar infiltrative process with calcified lymphadenopathy leading to narrowing of lobar bronchi and pulmonary arteries. An echocardiogram revealed pulmonary hypertension. Bronchoscopy showed left vocal cord paralysis and significant narrowing of the bilateral bronchi with mucosal thickening and multiple nodules. Transbronchial biopsy was compatible with sarcoidosis. Despite balloon angioplasty of the left lower lobe and pulmonary artery, and medical therapy with oral corticosteroids, her symptoms did not significantly improve. To the authors’ knowledge, the present report describes the first case of pulmonary sarcoidosis resulting in major airway, vascular and nerve compromise due to compressive lymphadenopathy and suspected concurrent granulomatous infiltration. Its presentation mimicked idiopathic mediastinal fibrosis.

  9. The European directive on renewable electricity: conflicts and compromises

    International Nuclear Information System (INIS)

    Rowlands, I.H.

    2005-01-01

    As part of its efforts to increase the use of renewable energy in Europe, a Directive regarding renewable electricity was agreed by the European Union in 2001. The purpose of this article is to examine this Directive, examining how the discussions surrounding its content unfolded. The investigation focuses upon three contentious issues that were debated during the Directive's development: the definition of 'renewable', the national targets for renewable electricity (their levels, as well as whether they should be 'binding' or 'indicative') and the questions associated with harmonisation (whether one Union-wide 'support scheme' for renewable electricity should be in place, and, if so, what it should be). During the 5 years that the Directive was negotiated, many intra-Union conflicts were eventually resolved, at least temporarily, by compromises. Nevertheless, some difficult decisions regarding the promotion of renewable electricity in the European Union still have to be taken

  10. Protecting Privacy of Shared Epidemiologic Data without Compromising Analysis Potential

    Directory of Open Access Journals (Sweden)

    John Cologne

    2012-01-01

    Full Text Available Objective. Ensuring privacy of research subjects when epidemiologic data are shared with outside collaborators involves masking (modifying the data, but overmasking can compromise utility (analysis potential. Methods of statistical disclosure control for protecting privacy may be impractical for individual researchers involved in small-scale collaborations. Methods. We investigated a simple approach based on measures of disclosure risk and analytical utility that are straightforward for epidemiologic researchers to derive. The method is illustrated using data from the Japanese Atomic-bomb Survivor population. Results. Masking by modest rounding did not adequately enhance security but rounding to remove several digits of relative accuracy effectively reduced the risk of identification without substantially reducing utility. Grouping or adding random noise led to noticeable bias. Conclusions. When sharing epidemiologic data, it is recommended that masking be performed using rounding. Specific treatment should be determined separately in individual situations after consideration of the disclosure risks and analysis needs.

  11. Managing Campus Energy: Compromising between Rapid Needs and Environmental Requirement

    Science.gov (United States)

    Ambariyanto, Ambariyanto; Utama, Yos J.; Purwanto

    2018-02-01

    The utilization of energy, especially electricity at Diponegoro University campus continues to increase in line with the development of the university. This increase has a direct impact on the increased costs to be paid by the university. Some of the causes of increased utilization of electrical energy is the construction of new buildings to meet the needs, increased learning activities and education, research activities in the laboratory, and various other activities. On the other hand, the increase of energy utilization is considered not good from the environment point of view, especially the utilization of electrical energy coming from non sustainable resources. Efforts to compromise on both are to develop policies in developing environmentally friendly buildings, efficiency in utilization of electrical energy, and development of sustainable energy sources.

  12. Thiamine absorption is not compromised in folate-deficient rats

    International Nuclear Information System (INIS)

    Walzem, R.L.; Clifford, A.J.

    1988-01-01

    Thiamine absorption and excretion were assessed in rats with severe folate deficiency (FD) by determining the fate of oral 3 H-labeled and intravenous 14 C-labeled thiamine over a 6-h test period. Thiamine status was evaluated in these same rats by measuring transketolase activity levels of blood before (TKA) and after (TPPE) addition of thiamine pyrophosphate to the incubation mixture of the assay procedure. Two additional experiments assessed active transport of thiamine and the effect of dietary succinylsulfathiazole (SST) on TKA and TPPE in rats with moderate FD. Intestinal absorption in general and thiamine absorption in particular and thiamine status were unaltered in rats with severe FD. Inanition associated with severe FD may impair thiamine status. Thiamine absorption by active transport was not compromised in FD, and dietary succinylsulfathiazole did not affect thiamine status

  13. Neglecting legumes has compromised human health and sustainable food production.

    Science.gov (United States)

    Foyer, Christine H; Lam, Hon-Ming; Nguyen, Henry T; Siddique, Kadambot H M; Varshney, Rajeev K; Colmer, Timothy D; Cowling, Wallace; Bramley, Helen; Mori, Trevor A; Hodgson, Jonathan M; Cooper, James W; Miller, Anthony J; Kunert, Karl; Vorster, Juan; Cullis, Christopher; Ozga, Jocelyn A; Wahlqvist, Mark L; Liang, Yan; Shou, Huixia; Shi, Kai; Yu, Jingquan; Fodor, Nandor; Kaiser, Brent N; Wong, Fuk-Ling; Valliyodan, Babu; Considine, Michael J

    2016-08-02

    The United Nations declared 2016 as the International Year of Pulses (grain legumes) under the banner 'nutritious seeds for a sustainable future'. A second green revolution is required to ensure food and nutritional security in the face of global climate change. Grain legumes provide an unparalleled solution to this problem because of their inherent capacity for symbiotic atmospheric nitrogen fixation, which provides economically sustainable advantages for farming. In addition, a legume-rich diet has health benefits for humans and livestock alike. However, grain legumes form only a minor part of most current human diets, and legume crops are greatly under-used. Food security and soil fertility could be significantly improved by greater grain legume usage and increased improvement of a range of grain legumes. The current lack of coordinated focus on grain legumes has compromised human health, nutritional security and sustainable food production.

  14. Addressing diversity in schools through dialogue and compromise

    DEFF Research Database (Denmark)

    Olsen, Tore Vincents

    2018-01-01

    This article evaluates a decentralized Danish model for dealing with cultural and religious diversity at individual schools. This evaluation is based upon normative theories of toleration, recognition and domination and examines whether the model implies compromise with the (liberal) educational...... values stipulated in the national legislation. The model, reconstructed from government publications, is based on reaching accommodation through dialogue between school staff and parents/students, with the pragmatic aim of facilitating the participation of students in everyday school activities....... The model is noteworthy because it appears to break with the widespread ‘retreat from multiculturalism’ predicated on the defence of liberal values, and because properly dealing with diversity at schools is important for ensuring students’ well-being and academic success....

  15. Enhanced susceptibility to predation in corals of compromised condition

    Directory of Open Access Journals (Sweden)

    Allan J. Bright

    2015-09-01

    Full Text Available The marine gastropod, Coralliophila abbreviata, is an obligate corallivore that causes substantial mortality in Caribbean Acropora spp. Considering the imperiled status of Acropora cervicornis and A. palmata, a better understanding of ecological interactions resulting in tissue loss may enable more effective conservation strategies. We examined differences in susceptibility of A. cervicornis to C. abbreviata predation based on coral tissue condition. Coral tissue condition was a strong determinant of snail prey choice, with snails preferring A. cervicornis fragments that were diseased or mechanically damaged over healthy fragments. In addition, snails always chose fragments undergoing active predation by another snail, while showing no preference for a non-feeding snail when compared with an undisturbed prey fragment. These results indicate that the condition of A. cervicornis prey influenced foraging behavior of C. abbreviata, creating a potential feedback that may exacerbate damage from predation in coral populations compromised by other types of disturbance.

  16. Enhanced susceptibility to predation in corals of compromised condition.

    Science.gov (United States)

    Bright, Allan J; Cameron, Caitlin M; Miller, Margaret W

    2015-01-01

    The marine gastropod, Coralliophila abbreviata, is an obligate corallivore that causes substantial mortality in Caribbean Acropora spp. Considering the imperiled status of Acropora cervicornis and A. palmata, a better understanding of ecological interactions resulting in tissue loss may enable more effective conservation strategies. We examined differences in susceptibility of A. cervicornis to C. abbreviata predation based on coral tissue condition. Coral tissue condition was a strong determinant of snail prey choice, with snails preferring A. cervicornis fragments that were diseased or mechanically damaged over healthy fragments. In addition, snails always chose fragments undergoing active predation by another snail, while showing no preference for a non-feeding snail when compared with an undisturbed prey fragment. These results indicate that the condition of A. cervicornis prey influenced foraging behavior of C. abbreviata, creating a potential feedback that may exacerbate damage from predation in coral populations compromised by other types of disturbance.

  17. Protecting Privacy of Shared Epidemiologic Data without Compromising Analysis Potential

    International Nuclear Information System (INIS)

    Cologne, J.; Nakashima, E.; Funamoto, S.; Grant, E.J.; Chen, Y.; Hiroaki Katayama, H.

    2012-01-01

    Objective. Ensuring privacy of research subjects when epidemiologic data are shared with outside collaborators involves masking (modifying) the data, but over masking can compromise utility (analysis potential). Methods of statistical disclosure control for protecting privacy may be impractical for individual researchers involved in small-scale collaborations. Methods. We investigated a simple approach based on measures of disclosure risk and analytical utility that are straightforward for epidemiologic researchers to derive. The method is illustrated using data from the Japanese Atomic-bomb Survivor population. Results. Masking by modest rounding did not adequately enhance security but rounding to remove several digits of relative accuracy effectively reduced the risk of identification without substantially reducing utility. Grouping or adding random noise led to noticeable bias. Conclusions. When sharing epidemiologic data, it is recommended that masking be performed using rounding. Specific treatment should be determined separately in individual situations after consideration of the disclosure risks and analysis needs

  18. Mitigating Reptile Road Mortality: Fence Failures Compromise Ecopassage Effectiveness

    Science.gov (United States)

    Baxter-Gilbert, James H.; Riley, Julia L.; Lesbarrères, David; Litzgus, Jacqueline D.

    2015-01-01

    Roadways pose serious threats to animal populations. The installation of roadway mitigation measures is becoming increasingly common, yet studies that rigorously evaluate the effectiveness of these conservation tools remain rare. A highway expansion project in Ontario, Canada included exclusion fencing and ecopassages as mitigation measures designed to offset detrimental effects to one of the most imperial groups of vertebrates, reptiles. Taking a multispecies approach, we used a Before-After-Control-Impact study design to compare reptile abundance on the highway before and after mitigation at an Impact site and a Control site from 1 May to 31 August in 2012 and 2013. During this time, radio telemetry, wildlife cameras, and an automated PIT-tag reading system were used to monitor reptile movements and use of ecopassages. Additionally, a willingness to utilize experiment was conducted to quantify turtle behavioral responses to ecopassages. We found no difference in abundance of turtles on the road between the un-mitigated and mitigated highways, and an increase in the percentage of both snakes and turtles detected dead on the road post-mitigation, suggesting that the fencing was not effective. Although ecopassages were used by reptiles, the number of crossings through ecopassages was lower than road-surface crossings. Furthermore, turtle willingness to use ecopassages was lower than that reported in previous arena studies, suggesting that effectiveness of ecopassages may be compromised when alternative crossing options are available (e.g., through holes in exclusion structures). Our rigorous evaluation of reptile roadway mitigation demonstrated that when exclusion structures fail, the effectiveness of population connectivity structures is compromised. Our project emphasizes the need to design mitigation measures with the biology and behavior of the target species in mind, to implement mitigation designs in a rigorous fashion, and quantitatively evaluate road

  19. Methamphetamine compromises gap junctional communication in astrocytes and neurons.

    Science.gov (United States)

    Castellano, Paul; Nwagbo, Chisom; Martinez, Luis R; Eugenin, Eliseo A

    2016-05-01

    Methamphetamine (meth) is a central nervous system (CNS) stimulant that results in psychological and physical dependency. The long-term effects of meth within the CNS include neuronal plasticity changes, blood-brain barrier compromise, inflammation, electrical dysfunction, neuronal/glial toxicity, and an increased risk to infectious diseases including HIV. Most of the reported meth effects in the CNS are related to dysregulation of chemical synapses by altering the release and uptake of neurotransmitters, especially dopamine, norepinephrine, and epinephrine. However, little is known about the effects of meth on connexin (Cx) containing channels, such as gap junctions (GJ) and hemichannels (HC). We examined the effects of meth on Cx expression, function, and its role in NeuroAIDS. We found that meth altered Cx expression and localization, decreased GJ communication between neurons and astrocytes, and induced the opening of Cx43/Cx36 HC. Furthermore, we found that these changes in GJ and HC induced by meth treatment were mediated by activation of dopamine receptors, suggesting that dysregulation of dopamine signaling induced by meth is essential for GJ and HC compromise. Meth-induced changes in GJ and HC contributed to amplified CNS toxicity by dysregulating glutamate metabolism and increasing the susceptibility of neurons and astrocytes to bystander apoptosis induced by HIV. Together, our results indicate that connexin containing channels, GJ and HC, are essential in the pathogenesis of meth and increase the sensitivity of the CNS to HIV CNS disease. Methamphetamine (meth) is an extremely addictive central nervous system stimulant. Meth reduced gap junctional (GJ) communication by inducing internalization of connexin-43 (Cx43) in astrocytes and reducing expression of Cx36 in neurons by a mechanism involving activation of dopamine receptors (see cartoon). Meth-induced changes in Cx containing channels increased extracellular levels of glutamate and resulted in higher

  20. Mitigating reptile road mortality: fence failures compromise ecopassage effectiveness.

    Directory of Open Access Journals (Sweden)

    James H Baxter-Gilbert

    Full Text Available Roadways pose serious threats to animal populations. The installation of roadway mitigation measures is becoming increasingly common, yet studies that rigorously evaluate the effectiveness of these conservation tools remain rare. A highway expansion project in Ontario, Canada included exclusion fencing and ecopassages as mitigation measures designed to offset detrimental effects to one of the most imperial groups of vertebrates, reptiles. Taking a multispecies approach, we used a Before-After-Control-Impact study design to compare reptile abundance on the highway before and after mitigation at an Impact site and a Control site from 1 May to 31 August in 2012 and 2013. During this time, radio telemetry, wildlife cameras, and an automated PIT-tag reading system were used to monitor reptile movements and use of ecopassages. Additionally, a willingness to utilize experiment was conducted to quantify turtle behavioral responses to ecopassages. We found no difference in abundance of turtles on the road between the un-mitigated and mitigated highways, and an increase in the percentage of both snakes and turtles detected dead on the road post-mitigation, suggesting that the fencing was not effective. Although ecopassages were used by reptiles, the number of crossings through ecopassages was lower than road-surface crossings. Furthermore, turtle willingness to use ecopassages was lower than that reported in previous arena studies, suggesting that effectiveness of ecopassages may be compromised when alternative crossing options are available (e.g., through holes in exclusion structures. Our rigorous evaluation of reptile roadway mitigation demonstrated that when exclusion structures fail, the effectiveness of population connectivity structures is compromised. Our project emphasizes the need to design mitigation measures with the biology and behavior of the target species in mind, to implement mitigation designs in a rigorous fashion, and quantitatively

  1. The Inflammatory Actions of Coagulant and Fibrinolytic Proteases in Disease

    Directory of Open Access Journals (Sweden)

    Michael Schuliga

    2015-01-01

    Full Text Available Aside from their role in hemostasis, coagulant and fibrinolytic proteases are important mediators of inflammation in diseases such as asthma, atherosclerosis, rheumatoid arthritis, and cancer. The blood circulating zymogens of these proteases enter damaged tissue as a consequence of vascular leak or rupture to become activated and contribute to extravascular coagulation or fibrinolysis. The coagulants, factor Xa (FXa, factor VIIa (FVIIa, tissue factor, and thrombin, also evoke cell-mediated actions on structural cells (e.g., fibroblasts and smooth muscle cells or inflammatory cells (e.g., macrophages via the proteolytic activation of protease-activated receptors (PARs. Plasmin, the principle enzymatic mediator of fibrinolysis, also forms toll-like receptor-4 (TLR-4 activating fibrin degradation products (FDPs and can release latent-matrix bound growth factors such as transforming growth factor-β (TGF-β. Furthermore, the proteases that convert plasminogen into plasmin (e.g., urokinase plasminogen activator evoke plasmin-independent proinflammatory actions involving coreceptor activation. Selectively targeting the receptor-mediated actions of hemostatic proteases is a strategy that may be used to treat inflammatory disease without the bleeding complications of conventional anticoagulant therapies. The mechanisms by which proteases of the coagulant and fibrinolytic systems contribute to extravascular inflammation in disease will be considered in this review.

  2. Characterizing Protease Specificity: How Many Substrates Do We Need?

    Directory of Open Access Journals (Sweden)

    Michael Schauperl

    Full Text Available Calculation of cleavage entropies allows to quantify, map and compare protease substrate specificity by an information entropy based approach. The metric intrinsically depends on the number of experimentally determined substrates (data points. Thus a statistical analysis of its numerical stability is crucial to estimate the systematic error made by estimating specificity based on a limited number of substrates. In this contribution, we show the mathematical basis for estimating the uncertainty in cleavage entropies. Sets of cleavage entropies are calculated using experimental cleavage data and modeled extreme cases. By analyzing the underlying mathematics and applying statistical tools, a linear dependence of the metric in respect to 1/n was found. This allows us to extrapolate the values to an infinite number of samples and to estimate the errors. Analyzing the errors, a minimum number of 30 substrates was found to be necessary to characterize substrate specificity, in terms of amino acid variability, for a protease (S4-S4' with an uncertainty of 5 percent. Therefore, we encourage experimental researchers in the protease field to record specificity profiles of novel proteases aiming to identify at least 30 peptide substrates of maximum sequence diversity. We expect a full characterization of protease specificity helpful to rationalize biological functions of proteases and to assist rational drug design.

  3. Pnserpin: A Novel Serine Protease Inhibitor from Extremophile Pyrobaculum neutrophilum

    Directory of Open Access Journals (Sweden)

    Huan Zhang

    2017-01-01

    Full Text Available Serine protease inhibitors (serpins are native inhibitors of serine proteases, constituting a large protein family with members spread over eukaryotes and prokaryotes. However, only very few prokaryotic serpins, especially from extremophiles, have been characterized to date. In this study, Pnserpin, a putative serine protease inhibitor from the thermophile Pyrobaculum neutrophilum, was overexpressed in Escherichia coli for purification and characterization. It irreversibly inhibits chymotrypsin-, trypsin-, elastase-, and subtilisin-like proteases in a temperature range from 20 to 100 °C in a concentration-dependent manner. The stoichiometry of inhibition (SI of Pnserpin for proteases decreases as the temperature increases, indicating that the inhibitory activity of Pnserpin increases with the temperature. SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis showed that Pnserpin inhibits proteases by forming a SDS-resistant covalent complex. Homology modeling and molecular dynamic simulations predicted that Pnserpin can form a stable common serpin fold. Results of the present work will help in understanding the structural and functional characteristics of thermophilic serpin and will broaden the current knowledge about serpins from extremophiles.

  4. Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

    International Nuclear Information System (INIS)

    Gomaa, O.M.; EI Shafey, H.M.

    2009-01-01

    Alkaline protease is one of the most important enzymes in industry, medicine, and research. In the present work, a comparative detection for alkaline protease activity was established for instant detection of enzyme activity. Eight different alkalophilic bacterial isolates were compared based on the clear zone they produced on skim milk agar. One strain gave an absolute clear zone in 16 hours and was used for alkaline protease detection. The result of Phenotypic identification using Biology Microlog 3 identified the isolate as Exiguobacterium acetylicum. The isolate under study showed slightly different characteristics from a known Exiguobacterium acetylicum strain. The isolate tolerated alkaline conditions up to ph 11, while good growth was evident at ph 7, the maximum alkaline protease activity was observed at ph 9 which reached up to 109.01 U/ml. The alkaline activity assay using alkaline protease enzyme assay were coordinating with those obtained by conductivity; there was a relevant decrease in conductivity at the maximum increase in enzyme activity, which proved the cell membrane conductivity has a close relation to alkaline protease production. This isolate has tolerated gamma radiation, the increase in dose (up to 4 Gy) gave wider clear zones in terms of diameter and this was relevant to the conductivity measurements

  5. Modelling of potentially promising SARS protease inhibitors

    International Nuclear Information System (INIS)

    Plewczynski, Dariusz; Hoffmann, Marcin; Grotthuss, Marcin von; Knizewski, Lukasz; Rychewski, Leszek; Eitner, Krystian; Ginalski, Krzysztof

    2007-01-01

    In many cases, at the beginning of a high throughput screening experiment some information about active molecules is already available. Active compounds (such as substrate analogues, natural products and inhibitors of related proteins) are often identified in low throughput validation studies on a biochemical target. Sometimes the additional structural information is also available from crystallographic studies on protein and ligand complexes. In addition, the structural or sequence similarity of various protein targets yields a novel possibility for drug discovery. Co-crystallized compounds from homologous proteins can be used to design leads for a new target without co-crystallized ligands. In this paper we evaluate how far such an approach can be used in a real drug campaign, with severe acute respiratory syndrome (SARS) coronavirus providing an example. Our method is able to construct small molecules as plausible inhibitors solely on the basis of the set of ligands from crystallized complexes of a protein target, and other proteins from its structurally homologous family. The accuracy and sensitivity of the method are estimated here by the subsequent use of an electronic high throughput screening flexible docking algorithm. The best performing ligands are then used for a very restrictive similarity search for potential inhibitors of the SARS protease within the million compounds from the Ligand.Info small molecule meta-database. The selected molecules can be passed on for further experimental validation

  6. Protease Production by Different Thermophilic Fungi

    Science.gov (United States)

    Macchione, Mariana M.; Merheb, Carolina W.; Gomes, Eleni; da Silva, Roberto

    A comparative study was carried out to evaluate protease production in solid-state fermentation (SSF) and submerged fermentation (SmF) by nine different thermophilic fungi — Thermoascus aurantiacus Miehe, Thermomyces lanuginosus, T. lanuginosus TO.03, Aspergillus flavus 1.2, Aspergillus sp. 13.33, Aspergillus sp. 13.34, Aspergillus sp. 13.35, Rhizomucor pusillus 13.36 and Rhizomucor sp. 13.37 — using substrates containing proteins to induce enzyme secretion. Soybean extract (soybean milk), soybean flour, milk powder, rice, and wheat bran were tested. The most satisfactory results were obtained when using wheat bran in SSF. The fungi that stood out in SSF were T. lanuginosus, T. lanuginosus TO.03, Aspergillus sp. 13.34, Aspergillus sp. 13.35, and Rhizomucor sp. 13.37, and those in SmF were T. aurantiacus, T. lanuginosus TO.03, and 13.37. In both fermentation systems, A. flavus 1.2 and R. pusillus 13.36 presented the lowest levels of proteolytic activity.

  7. Antibody proteases: induction of catalytic response.

    Science.gov (United States)

    Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D

    2002-10-01

    Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.

  8. Modelling of potentially promising SARS protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

    Plewczynski, Dariusz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Hoffmann, Marcin [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Grotthuss, Marcin von [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Knizewski, Lukasz [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland); Rychewski, Leszek [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Eitner, Krystian [BioInfoBank Institute, Limanowskiego 24A/16, 60-744 Poznan (Poland); Ginalski, Krzysztof [Interdisciplinary Centre for Mathematical and Computational Modelling, ICM, Warsaw University, Pawinskiego 5a Street, 02-106 Warsaw (Poland)

    2007-07-18

    In many cases, at the beginning of a high throughput screening experiment some information about active molecules is already available. Active compounds (such as substrate analogues, natural products and inhibitors of related proteins) are often identified in low throughput validation studies on a biochemical target. Sometimes the additional structural information is also available from crystallographic studies on protein and ligand complexes. In addition, the structural or sequence similarity of various protein targets yields a novel possibility for drug discovery. Co-crystallized compounds from homologous proteins can be used to design leads for a new target without co-crystallized ligands. In this paper we evaluate how far such an approach can be used in a real drug campaign, with severe acute respiratory syndrome (SARS) coronavirus providing an example. Our method is able to construct small molecules as plausible inhibitors solely on the basis of the set of ligands from crystallized complexes of a protein target, and other proteins from its structurally homologous family. The accuracy and sensitivity of the method are estimated here by the subsequent use of an electronic high throughput screening flexible docking algorithm. The best performing ligands are then used for a very restrictive similarity search for potential inhibitors of the SARS protease within the million compounds from the Ligand.Info small molecule meta-database. The selected molecules can be passed on for further experimental validation.

  9. Identification of Cysteine Proteases and Screening of Cysteine Protease Inhibitors in Biological Samples by a Two-Dimensional Gel System of Zymography and Reverse Zymography

    OpenAIRE

    Saitoh, Eiichi; Yamamoto, Shinya; Okamoto, Eishiro; Hayakawa, Yoshimi; Hoshino, Takashi; Sato, Ritsuko; Isemura, Satoko; Ohtsubo, Sadami; Taniguchi, Masayuki

    2007-01-01

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the fi rst-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic...

  10. 11 Soil Microbial Biomass

    African Journals Online (AJOL)

    186–198. Insam H. (1990). Are the soil microbial biomass and basal respiration governed by the climatic regime? Soil. Biol. Biochem. 22: 525–532. Insam H. D. and Domsch K. H. (1989). Influence of microclimate on soil microbial biomass. Soil Biol. Biochem. 21: 211–21. Jenkinson D. S. (1988). Determination of microbial.

  11. Molecular microbial ecology manual

    NARCIS (Netherlands)

    Kowalchuk, G.A.; Bruijn, de F.J.; Head, I.M.; Akkermans, A.D.L.

    2004-01-01

    The field of microbial ecology has been revolutionized in the past two decades by the introduction of molecular methods into the toolbox of the microbial ecologist. This molecular arsenal has helped to unveil the enormity of microbial diversity across the breadth of the earth's ecosystems, and has

  12. Microbial Rechargeable Battery

    NARCIS (Netherlands)

    Molenaar, Sam D.; Mol, Annemerel R.; Sleutels, Tom H.J.A.; Heijne, Ter Annemiek; Buisman, Cees J.N.

    2016-01-01

    Bioelectrochemical systems hold potential for both conversion of electricity into chemicals through microbial electrosynthesis (MES) and the provision of electrical power by oxidation of organics using microbial fuel cells (MFCs). This study provides a proof of concept for a microbial

  13. Childhood microbial keratitis

    Directory of Open Access Journals (Sweden)

    Abdullah G Al Otaibi

    2012-01-01

    Conclusion: Children with suspected microbial keratitis require comprehensive evaluation and management. Early recognition, identifying the predisposing factors and etiological microbial organisms, and instituting appropriate treatment measures have a crucial role in outcome. Ocular trauma was the leading cause of childhood microbial keratitis in our study.

  14. Production of arapaima protein hydrolysate using Aspergillus flavo-furcatis protease and pancreatin

    Directory of Open Access Journals (Sweden)

    Flávia de Carvalho Paiva

    2015-03-01

    Full Text Available The processing of arapaima (Arapaima gigas generates a lot of residues that can be used for the development of new products of industrial interest. This study aimed at evaluating the production of protein hydrolysates from arapaima residues using Aspergillus flavo-furcatis protease and commercial pancreatin, as well as characterizing their nutritional and microbiological qualities. The raw material used was meat mechanically separated from arapaima carcasses (MMSA. Two products were developed: a protein hydrolysate of arapaima using a commercial enzyme (PHACE and another one using microbial enzyme (PHAME. The MMSA and the hydrolysates were analyzed for chemical composition, microbiological quality, degree of hydrolysis, digestibility and amino acid profile. The results showed that the PHACE protein content was 73.47 %. This value was significantly higher, when compared to the PHAME (58.03 %. However, both products showed high digestibility values, absence of microbial contaminants and reduced lipid content. Among the enzymes used, pancreatin was the most efficient one in the preparation of the final product, which showed essential amino acids content higher than the requirements for human adults. The hydrolysate developed using A. flavo-furcatis enzymes presented essential amino acids score lower than 1.0, being tryptophan the most limiting one.

  15. Sustainable Development Compromise[d] in the Planning of Metro Vancouver’s Agricultural Lands—the Jackson Farm Case

    Directory of Open Access Journals (Sweden)

    Meg Holden

    2013-11-01

    Full Text Available This research provides analysis of the case of the Jackson Farm development application, embedded within the particular dynamics of the municipal, regional, and provincial sustainability land use policy culture of the Metro Vancouver region, in Canada. Within a culture of appreciation of the increasing need for sustainability in land use policy, including the protection of agricultural lands at the provincial level through the Agricultural Land Reserve (ALR, to urban intensification and protection of the green zone at the regional scale, lies a political conflict that comes into focus in individual land use decisions, within municipalities struggling for autonomy. This case is neither driven strictly by “the politics of the highest bidder” nor by policy failure; the case of the Jackson Farm is instead a case of the challenges of implementing inter-governmental coordination and collaborative governance in a context of both significant sustainability policy and urban growth. The process can be seen to follow an ecological modernization agenda, seeking “win–win” alternatives rather than recognizing that typical compromises, over time, may tip the direction of development away from sustainability policy goals. Understanding the twists, turns, and eventual compromise reached in the case of the Jackson Farm brings to light the implications of the shift in the regional planning culture which may necessitate a less flexible, more structured prioritization of competing goals within plans and policies in order to meet sustainability goals. We highlight this, and present an alternative implementation process within the existing policy regime with potential to aid the specific goal of agricultural land protection.

  16. Identification of an archaeal presenilin-like intramembrane protease.

    Science.gov (United States)

    Torres-Arancivia, Celia; Ross, Carolyn M; Chavez, Jose; Assur, Zahra; Dolios, Georgia; Mancia, Filippo; Ubarretxena-Belandia, Iban

    2010-09-29

    The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ) implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs). The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP) without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

  17. Identification of an archaeal presenilin-like intramembrane protease.

    Directory of Open Access Journals (Sweden)

    Celia Torres-Arancivia

    Full Text Available BACKGROUND: The GXGD-type diaspartyl intramembrane protease, presenilin, constitutes the catalytic core of the γ-secretase multi-protein complex responsible for activating critical signaling cascades during development and for the production of β-amyloid peptides (Aβ implicated in Alzheimer's disease. The only other known GXGD-type diaspartyl intramembrane proteases are the eukaryotic signal peptide peptidases (SPPs. The presence of presenilin-like enzymes outside eukaryots has not been demonstrated. Here we report the existence of presenilin-like GXGD-type diaspartyl intramembrane proteases in archaea. METHODOLOGY AND PRINCIPAL FINDINGS: We have employed in vitro activity assays to show that MCMJR1, a polytopic membrane protein from the archaeon Methanoculleus marisnigri JR1, is an intramembrane protease bearing the signature YD and GXGD catalytic motifs of presenilin-like enzymes. Mass spectrometry analysis showed MCMJR1 could cleave model intramembrane protease substrates at several sites within their transmembrane region. Remarkably, MCMJR1 could also cleave substrates derived from the β-amyloid precursor protein (APP without the need of protein co-factors, as required by presenilin. Two distinct cleavage sites within the transmembrane domain of APP could be identified, one of which coincided with Aβ40, the predominant site processed by γ-secretase. Finally, an established presenilin and SPP transition-state analog inhibitor could inhibit MCMJR1. CONCLUSIONS AND SIGNIFICANCE: Our findings suggest that a primitive GXGD-type diaspartyl intramembrane protease from archaea can recapitulate key biochemical properties of eukaryotic presenilins and SPPs. MCMJR1 promises to be a more tractable, simpler system for in depth structural and mechanistic studies of GXGD-type diaspartyl intramembrane proteases.

  18. Taking a Bad Turn: Compromised DNA Damage Response in Leukemia

    Directory of Open Access Journals (Sweden)

    Nadine Nilles

    2017-05-01

    Full Text Available Genomic integrity is of outmost importance for the survival at the cellular and the organismal level and key to human health. To ensure the integrity of their DNA, cells have evolved maintenance programs collectively known as the DNA damage response. Particularly challenging for genome integrity are DNA double-strand breaks (DSB and defects in their repair are often associated with human disease, including leukemia. Defective DSB repair may not only be disease-causing, but further contribute to poor treatment outcome and poor prognosis in leukemia. Here, we review current insight into altered DSB repair mechanisms identified in leukemia. While DSB repair is somewhat compromised in all leukemic subtypes, certain key players of DSB repair are particularly targeted: DNA-dependent protein kinase (DNA-PK and Ku70/80 in the non-homologous end-joining pathway, as well as Rad51 and breast cancer 1/2 (BRCA1/2, key players in homologous recombination. Defects in leukemia-related DSB repair may not only arise from dysfunctional repair components, but also indirectly from mutations in key regulators of gene expression and/or chromatin structure, such as p53, the Kirsten ras oncogene (K-RAS, and isocitrate dehydrogenase 1 and 2 (IDH1/2. A detailed understanding of the basis for defective DNA damage response (DDR mechanisms for each leukemia subtype may allow to further develop new treatment methods to improve treatment outcome and prognosis for patients.

  19. Tracheostomy in neurologically compromised paediatric patients: role of starplasty.

    Science.gov (United States)

    Gupta, A; Stokken, J; Krakovitz, P; Malhotra, P; Anne, S

    2015-10-01

    Starplasty tracheostomy is an alternative to traditional tracheostomy. This paper reviews neurologically compromised paediatric patients with tracheostomies and discusses the role of starplasty tracheostomy. A retrospective review was conducted of paediatric patients with a neurological disorder who underwent tracheostomy between 1997 and 2011. Forty-eight patients, with an average age of 7.3 years, were identified. The most common indications for tracheostomy were: ventilator dependence (39.6 per cent), an inability to tolerate secretions or recurrent aspiration pneumonia (33.3 per cent), and upper respiratory obstruction or hypotonia (12.5 per cent). The most common underlying neurological diagnosis was cerebral palsy. There were no early complications. Eighteen (43 per cent) of 42 patients with follow up experienced at least 1 delayed complication. Only 12 patients (28.6 per cent) were decannulated. Patients with primary neurological diagnoses have low rates of decannulation; starplasty tracheostomy should be considered for these patients. Patients with seizure disorder or acute neurological injury tended to have a higher short-term decannulation rate; traditional tracheostomy is recommended in these patients.

  20. Compromised careers: the occupational transition of immigration and resettlement.

    Science.gov (United States)

    Suto, Melinda

    2009-01-01

    Work is a significant occupational transition that occurs with immigration and resettlement. Problems finding work and regaining economic capital are multi-factorial, differentiated by gender and mediated by specific contexts. Surprisingly, past education and work experience are unreliable predictors of successful employment outcomes. Critical theory and ethnographic concepts informed the methodological approach. Data were generated primarily through in-depth interviews, conducted in English, with 14 well-educated women who immigrated to Canada as adults and sought employment in their professions. The thematic findings were analyzed using Bourdieu's [7] concepts of capital, field and habitus. The theme Compromised Careers describes the downward occupational (work) mobility that occurs despite expectations that education, credentials and work experience are transferable to desirable employment. A devaluation of foreign qualifications and no relevant Canadian work experience function with gendered responsibilities, less social support, and time spent in resettlement activities to create negative work trajectories. The role that federal policies and professional organizations play is examined to reveal the tension between individuals' efforts to find employment and institutional barriers that impede these actions. A critical inquiry approach examined the ruling relations to show how power and privilege function in relation to migrants' occupational transitions.

  1. Towards mesoscience the principle of compromise in competition

    CERN Document Server

    Li, Jinghai

    2014-01-01

    This brief is devoted to providing a complete outline of meso-science by briefing the relevant contents from the published book and by updating evidences and concepts of meso-science. The importance of meso-science in solving various problems in energy, resource, and the environment is introduced.  The whole evolutionary development of the EMMS principle is reviewed to show how a simple idea on the customized modeling of particle clustering in gas-solid systems was developed, verified, extended, and finally generalized into the common principle of compromise in competition between dominant mechanisms for all mesoscale phenomena in science and engineering, leading to the proposition of meso-science. More importantly, updates on the concept of meso-science and perspectives are presented, along with new insights and findings from after the publication of the original book. In this way, we hope to help readers more easily familiarize themselves with meso-science, and to trigger interest and attention to this int...

  2. Auditing Litigation and Claims: Conflicts and the Compromise of Privilege

    Directory of Open Access Journals (Sweden)

    Harleen Kaur

    2013-12-01

    Full Text Available Auditing standards require an auditor to make various enquiries about liabilities in general this may entail consideration of potential litigations and claims that the audited entity may be facing. To perform this part of audit, the auditors will generally seek representation letters from lawyers of the company detailing an estimate prepared by management, confirmed by their lawyers through a representation letter, and then sent directly to the auditors. This paper reviews the implications for the auditing profession of a case that involved auditors seeking such representation letters. The case involves litigation between theWestpac Banking Corporation and 789TEN Pty Ltd. While theWestpac case confirmed the legal position of the auditor in their task of collecting evidence in order to form an opinion in Australia, it highlights a significant anomaly under the law and should place the issue of solicitor’s representation letters as audit evidence firmly on the agenda of policymakers. This issue of the compromise of legal privilege during the conduct of an audit is also not confined to Australia: other common law jurisdictions, such as the UK and the US, have also sought to clarify the position of auditors when issues of the integrity of legal privacy privilege arise.

  3. Diversity of both the cultivable protease-producing bacteria and bacterial extracellular proteases in the coastal sediments of King George Island, Antarctica.

    Directory of Open Access Journals (Sweden)

    Ming-Yang Zhou

    Full Text Available Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 10(5 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%, Flavobacterium (21.0% and Lacinutrix (16.2%. Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea.

  4. Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases.

    Science.gov (United States)

    Luo, Jie; Xiao, Chen; Zhang, Hao; Ren, Fazheng; Lei, Xingen; Yang, Zibiao; Yu, Zhengquan

    2018-04-01

    The herbaceous plant Cynanchum otophyllum Schneid. is widely used as a milk coagulant to make a Chinese traditional milk product, milk cake. However, the milk-clotting compounds and their mechanism remain unclear. In this study, crude proteases were extracted from the dried leaves of Cynanchum otophyllum Schneid. using citric acid-phosphate buffer and then partially purified by weak anion exchange chromatography. Two proteases, QA and QC, with molecular weights of 14 and 27 kDa, respectively, were shown to exhibit milk-clotting activity. A study of the effects of pH and temperature on the milk-clotting activity and proteolytic activity of the proteases showed that they exhibited good pH stability from pH 5.5 to 7.5 and good thermal stability at temperatures from 50 to 70°C. The QA and QC were the cysteine proteases, able to hydrolyze β-casein and κ-casein completely, and α-casein partially. The cleavage site on κ-casein determined by Orbitrap (Thermo Fisher Scientific, San Jose, CA) analysis showed that QA and QC could cleave κ-casein at Ser132-Thr133. Overall, the results suggest that the Cynanchum otophyllum Schneid. proteases are a promising milk-clotting enzyme that could be used for manufacturing milk cake and cheese. Copyright © 2018 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  5. Cysteine Protease (Capparin from Capsules of Caper (Capparis spinosa

    Directory of Open Access Journals (Sweden)

    Yasar Demir

    2008-01-01

    Full Text Available Proteases are enzymes that perform very important functions in organisms and are used for a variety of objectives in vitro. In recent years, proteases have been used for clinical, pharmaceutical (alimentary digestion, anti-inflammatory, etc. and industrial applications (cheese production, meat tenderizing, leather tanning. In this research, a protease has been purified from capsules of caper (Capparis spinosa and characterized. Caper plants have been used for food and medicine since ancient times. The plant grows abundantly in certain regions of Turkey. Ammonium sulphate fractionation and a CM Sephadex column were used for purification of the enzyme. The purification enzyme has an optimum pH=5.0 and its optimum temperature was 60 °C. The vmax and Km values determined by Lineweaver-Burk graphics were 1.38 μg/(L·min and 0.88 μg/L, respectively. The purification degree and the molecular mass of the enzyme (46 kDa were determined by SDS-PAGE and gel filtration chromatography. It was investigated whether the purified and characterized protease could cause milk to congeal or digest chicken and cow meat. The results show that protease can be used for industrial production.

  6. Detection of protease activity in cells and animals.

    Science.gov (United States)

    Verdoes, Martijn; Verhelst, Steven H L

    2016-01-01

    Proteases are involved in a wide variety of biologically and medically important events. They are entangled in a complex network of processes that regulate their activity, which makes their study intriguing, but challenging. For comprehensive understanding of protease biology and effective drug discovery, it is therefore essential to study proteases in models that are close to their complex native environments such as live cells or whole organisms. Protease activity can be detected by reporter substrates and activity-based probes, but not all of these reagents are suitable for intracellular or in vivo use. This review focuses on the detection of proteases in cells and in vivo. We summarize the use of probes and substrates as molecular tools, discuss strategies to deliver these tools inside cells, and describe sophisticated read-out techniques such as mass spectrometry and various imaging applications. This article is part of a Special Issue entitled: Physiological Enzymology and Protein Functions. Copyright © 2015 Elsevier B.V. All rights reserved.

  7. Synthesis of glycinamides using protease immobilized magnetic nanoparticles

    Directory of Open Access Journals (Sweden)

    Abha Sahu

    2016-12-01

    Full Text Available In the present investigation, Bacillus subtilis was isolated from slaughterhouse waste and screened for the production of protease enzyme. The purified protease was successfully immobilized on magnetic nanoparticles (MNPs and used for the synthesis of series of glycinamides. The binding and thermal stability of protease on MNPs was confirmed by FTIR spectroscopy and TGA analysis. The surface morphology of MNPs before and after protease immobilization was carried out using SEM analysis. XRD pattern revealed no phase change in MNPs after enzyme immobilization. The processing parameters for glycinamides synthesis viz. temperature, pH, and time were optimized using Response Surface Methodology (RSM by using Design Expert (9.0.6.2. The maximum yield of various amides 2 butyramidoacetic acid (AMD-1,83.4%, 2-benzamidoacetic acid (AMD-2,80.5% and 2,2′((carboxymethyl amino-2-oxoethyl-2-hydroxysuccinylbis(azanediyldiacetic acid (AMD-3,80.8% formed was observed at pH-8, 50 °C and 30 min. The synthesized immobilized protease retained 70% of the initial activity even after 8 cycles of reuse.

  8. Characterization and Potential Use of Cuttlefish Skin Gelatin Hydrolysates Prepared by Different Microbial Proteases

    Directory of Open Access Journals (Sweden)

    Mourad Jridi

    2014-01-01

    Full Text Available Composition, functional properties, and in vitro antioxidant activities of gelatin hydrolysates prepared from cuttlefish skin were investigated. Cuttlefish skin gelatin hydrolysates (CSGHs were obtained by treatment with crude enzyme preparations from Bacillus licheniformis NH1, Bacillus mojavensis A21, Bacillus subtilis A26, and commercial alcalase. All CSGHs had high protein contents, 74.3–78.3%, and showed excellent solubility (over 90%. CSGH obtained by alcalase demonstrated high antioxidant activities monitored by β-carotene bleaching, DPPH radical scavenging, lipid peroxidation inhibition, and reducing power activity. Its antioxidant activity remained stable or increased in a wide range of pH (1–9, during heating treatment (100°C for 240 min and after gastrointestinal digestion simulation. In addition, alcalase-CSGH was incorporated into turkey meat sausage to determine its effect on lipid oxidation during 35 days of storage period. At 0.5 mg/g, alcalase-CSGH delayed lipid oxidation monitored by TBARS and conjugated diene up to 10 days compared to vitamin C. The results reveal that CSGHs could be used as food additives possessing both antioxidant activity and functional properties.

  9. Microbial Biosensor for the Detection of Protease-Virulent Factors from Pathogens

    Science.gov (United States)

    2017-04-28

    pathogen signalling molecules. With that goal in mind , researchers have developed various types of biosensors that detect infectious determinants...life to boost their capability for rapid determination of clean water sources during field deployment. Despite the promising results of these studies

  10. Evaluation of a D-amino-acid-containing fluorescence resonance energy transfer peptide library for profiling prokaryotic proteases

    NARCIS (Netherlands)

    Kaman, W.E.; Voskamp-Visser, I.; de Jongh, D.M.C.; Endtz, H.P.; van Belkum, A.; Hays, J.P.; Bikker, F.J.

    2013-01-01

    Bacterial proteases play an important role in a broad spectrum of processes, including colonization, proliferation, and virulence. In this respect, bacterial proteases are potential biomarkers for bacterial diagnosis and targets for novel therapeutic protease inhibitors. To investigate these

  11. The Cysteine Protease–Cysteine Protease Inhibitor System Explored in Soybean Nodule Development

    Directory of Open Access Journals (Sweden)

    Marian Dorcas Quain

    2013-08-01

    Full Text Available Almost all protease families have been associated with plant development, particularly senescence, which is the final developmental stage of every organ before cell death. Proteolysis remobilizes and recycles nitrogen from senescent organs that is required, for example, seed development. Senescence-associated expression of proteases has recently been characterized using large-scale gene expression analysis seeking to identify and characterize senescence-related genes. Increasing activities of proteolytic enzymes, particularly cysteine proteases, are observed during the senescence of legume nodules, in which a symbiotic relationship between the host plant and bacteria (Rhizobia facilitate the fixation of atmospheric nitrogen. It is generally considered that cysteine proteases are compartmentalized to prevent uncontrolled proteolysis in nitrogen-fixing nodules. In addition, the activities of cysteine proteases are regulated by endogenous cysteine protease inhibitors called cystatins. These small proteins form reversible complexes with cysteine proteases, leading to inactivation. However, very little is currently known about how the cysteine protease-cysteine protease inhibitor (cystatin system is regulated during nodule development. Moreover, our current understanding of the expression and functions of proteases and protease inhibitors in nodules is fragmented. To address this issue, we have summarized the current knowledge and techniques used for studying proteases and their inhibitors including the application of “omics” tools, with a particular focus on changes in the cysteine protease-cystatin system during nodule development.

  12. Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS.

    Science.gov (United States)

    Neustadt, Madlen; Costina, Victor; Kupfahl, Claudio; Buchheidt, Dieter; Eckerskorn, Christoph; Neumaier, Michael; Findeisen, Peter

    2009-06-01

    Early diagnosis of life-threatening invasive aspergillosis in neutropenic patients remains challenging because current laboratory methods have limited diagnostic sensitivity and/or specificity. Aspergillus species are known to secrete various pathogenetically relevant proteases and the monitoring of their protease activity in serum specimens might serve as a new diagnostic approach.For the characterization and identification of secreted proteases, the culture supernatant of Aspergillus fumigatus was fractionated using free flow electrophoresis (Becton Dickinson). Protease activity of separated fractions was measured using fluorescently labeled reporter peptides. Fractions were also co-incubated in parallel with various protease inhibitors that specifically inhibit a distinct class of proteases e.g. metallo- or cysteine-proteases. Those fractions with high protease activity were further subjected to LC-MS/MS analysis for protease identification. The highest protease activity was measured in fractions with an acidic pH range. The results of the 'inhibitor-panel' gave a clear indication that it is mainly metallo- and serine-proteases that are involved in the degradation of reporter peptides. Furthermore, several proteases were identified that facilitate the optimization of reporter peptides for functional protease profiling as a diagnostic tool for invasive aspergillosis.

  13. Capacity for Preferences: Respecting Patients with Compromised Decision-Making.

    Science.gov (United States)

    Wasserman, Jason Adam; Navin, Mark Christopher

    2018-05-01

    When a patient lacks decision-making capacity, then according to standard clinical ethics practice in the United States, the health care team should seek guidance from a surrogate decision-maker, either previously selected by the patient or appointed by the courts. If there are no surrogates willing or able to exercise substituted judgment, then the team is to choose interventions that promote a patient's best interests. We argue that, even when there is input from a surrogate, patient preferences should be an additional source of guidance for decisions about patients who lack decision-making capacity. Our proposal builds on other efforts to help patients who lack decision-making capacity provide input into decisions about their care. For example, "supported," "assisted," or "guided" decision-making models reflect a commitment to humanistic patient engagement and create a more supportive process for patients, families, and health care teams. But often, they are supportive processes for guiding a patient toward a decision that the surrogate or team believes to be in the patient's medical best interests. Another approach holds that taking seriously the preferences of such a patient can help surrogates develop a better account of what the patient's treatment choices would have been if the patient had retained decision-making capacity; the surrogate then must try to integrate features of the patient's formerly rational self with the preferences of the patient's currently compromised self. Patients who lack decision-making capacity are well served by these efforts to solicit and use their preferences to promote best interests or to craft would-be autonomous patient images for use by surrogates. However, we go further: the moral reasons for valuing the preferences of patients without decision-making capacity are not reducible to either best-interests or (surrogate) autonomy considerations but can be grounded in the values of liberty and respect for persons. This has

  14. Chemical Tools for the Study of Intramembrane Proteases.

    Science.gov (United States)

    Nguyen, Minh T N; Van Kersavond, Tim; Verhelst, Steven H L

    2015-11-20

    Intramembrane proteases (IMPs) reside inside lipid bilayers and perform peptide hydrolysis in transmembrane or juxtamembrane regions of their substrates. Many IMPs are involved in crucial regulatory pathways and human diseases, including Alzheimer's disease, Parkinson's disease, and diabetes. In the past, chemical tools have been instrumental in the study of soluble proteases, enabling biochemical and biomedical research in complex environments such as tissue lysates or living cells. However, IMPs place special challenges on probe design and applications, and progress has been much slower than for soluble proteases. In this review, we will give an overview of the available chemical tools for IMPs, including activity-based probes, affinity-based probes, and synthetic substrates. We will discuss how these have been used to increase our structural and functional understanding of this fascinating group of enzymes, and how they might be applied to address future questions and challenges.

  15. Effects of protease inhibitors on radiation transformation in vitro

    International Nuclear Information System (INIS)

    Kennedy, A.R.; Little, J.B.

    1981-01-01

    We have investigated the effects of three protease inhibitors, antipain, leupeptin, and soybean trypsin inhibitor, on the induction of oncogenic transformation in mouse C3H10T 1/2 cells by X-rays. The patterns of inhibition by the three protease inhibitors were different. Antipain was the most effective, having the ability to suppress completely radiation transformation as well as radiation transformation enhanced by the phorbol ester promoting agent 12-O-tetradecanoylphorbol-13-acetate. The fact that antipain could suppress transformation when present for only 1 day following irradiation suggests that an effect on a DNA repair process might be important in its action. Leupeptin was less effective than antipain in its inhibition of radiation transformation. Soybean trypsin inhibitor suppressed only the promotional effects of 12-O-tetradecanoylphorbol-13-acetate on transformation. Our results suggest that there may be more than one protease involved in carcinogenesis

  16. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  17. Purification and characterization of alkaline proteases from aspergillus terreus

    International Nuclear Information System (INIS)

    Hussain, A.; Mannan, A.; Zubair, H.; Mirza, B.

    2010-01-01

    Proteases belong to an important class of enzymes known as hydrolases and catalyze hydrolysis of proteins. They act primarily to degrade proteins that are used for energy production and as biosynthetic precursors. In the following study, protease produced from Aspergillus terreus was found to be thermo stable and included in the category of alkaline serine and metallo protease. During partial purification, presence of enzyme in 60% (NH/sub 4/)/sub 2/SO/sub 4/ indicated small molecular weight polypeptide; later purification with Sephadex G-75 fractionation yielded a single proteolytic active molecule. At final purification step, the increase in specific activity of the enzyme was 7.5 fold with 23% yield. SDS-PAGE analysis revealed that alkaline protease of Aspergillus terreus is a monomer with approximate molecular weight of 35 kDa. Optimum pH for protease activity was found in the range of 7.5-11.0 (maximum at pH 8.5), thus apparently classified as an alkaline protease. The enzyme was thermo stable towards high temperature (60 deg. C), however it denatured irreversibly at 70 deg. C showing 80% loss of activity. The maximum proteolytic activity was found at 40 deg. C. The enzyme was effectively inhibited by PMSF, EDTA and urea whereas iodoacetamide and thiourea did not result in any loss in activity while cysteine was found to be activator molecule. The study with metal ions Mg/sup +2/, Mn/sup +2/ and Fe/sup +3/ (1 mM each) showed minute stimulatory effects on enzyme activity. Co/sup +2/ and Ca/sup +2/ (1 mM) had neither excitatory nor inhibitory effect while Hg/sup +2/ and Cu/sup +2/ (1 mM) slightly reduced the enzyme activity. (author)

  18. m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration.

    Science.gov (United States)

    Patron, Maria; Sprenger, Hans-Georg; Langer, Thomas

    2018-03-01

    The function of mitochondria depends on ubiquitously expressed and evolutionary conserved m-AAA proteases in the inner membrane. These ATP-dependent peptidases form hexameric complexes built up of homologous subunits. AFG3L2 subunits assemble either into homo-oligomeric isoenzymes or with SPG7 (paraplegin) subunits into hetero-oligomeric proteolytic complexes. Mutations in AFG3L2 are associated with dominant spinocerebellar ataxia (SCA28) characterized by the loss of Purkinje cells, whereas mutations in SPG7 cause a recessive form of hereditary spastic paraplegia (HSP7) with motor neurons of the cortico-spinal tract being predominantly affected. Pleiotropic functions have been assigned to m-AAA proteases, which act as quality control and regulatory enzymes in mitochondria. Loss of m-AAA proteases affects mitochondrial protein synthesis and respiration and leads to mitochondrial fragmentation and deficiencies in the axonal transport of mitochondria. Moreover m-AAA proteases regulate the assembly of the mitochondrial calcium uniporter (MCU) complex. Impaired degradation of the MCU subunit EMRE in AFG3L2-deficient mitochondria results in the formation of deregulated MCU complexes, increased mitochondrial calcium uptake and increased vulnerability of neurons for calcium-induced cell death. A reduction of calcium influx into the cytosol of Purkinje cells rescues ataxia in an AFG3L2-deficient mouse model. In this review, we discuss the relationship between the m-AAA protease and mitochondrial calcium homeostasis and its relevance for neurodegeneration and describe a novel mouse model lacking MCU specifically in Purkinje cells. Our results pledge for a novel view on m-AAA proteases that integrates their pleiotropic functions in mitochondria to explain the pathogenesis of associated neurodegenerative disorders.

  19. Human eosinophils constitutively express a unique serine protease, PRSS33.

    Science.gov (United States)

    Toyama, Sumika; Okada, Naoko; Matsuda, Akio; Morita, Hideaki; Saito, Hirohisa; Fujisawa, Takao; Nakae, Susumu; Karasuyama, Hajime; Matsumoto, Kenji

    2017-07-01

    Eosinophils play important roles in asthma, especially airway remodeling, by producing various granule proteins, chemical mediators, cytokines, chemokines and proteases. However, protease production by eosinophils is not fully understood. In the present study, we investigated the production of eosinophil-specific proteases/proteinases by transcriptome analysis. Human eosinophils and other cells were purified from peripheral blood by density gradient sedimentation and negative/positive selections using immunomagnetic beads. Protease/proteinase expression in eosinophils and release into the supernatant were evaluated by microarray analysis, qPCR, ELISA, flow cytometry and immunofluorescence staining before and after stimulation with eosinophil-activating cytokines and secretagogues. mRNAs for extracellular matrix proteins in human normal fibroblasts were measured by qPCR after exposure to recombinant protease serine 33 (PRSS33) protein (rPRSS33), created with a baculovirus system. Human eosinophils expressed relatively high levels of mRNA for metalloproteinase 25 (MMP25), a disintegrin and metalloprotease 8 (ADAM8), ADAM10, ADAM19 and PRSS33. Expression of PRSS33 was the highest and eosinophil-specific. PRSS33 mRNA expression was not affected by eosinophil-activating cytokines. Immunofluorescence staining showed that PRSS33 was co-localized with an eosinophil granule protein. PRSS33 was not detected in the culture supernatant of eosinophils even after stimulation with secretagogues, but its cell surface expression was increased. rPRSS33 stimulation of human fibroblasts increased expression of collagen and fibronectin mRNAs, at least in part via protease-activated receptor-2 activation. Activated eosinophils may induce fibroblast extracellular matrix protein synthesis via cell surface expression of PRSS33, which would at least partly explain eosinophils' role(s) in airway remodeling. Copyright © 2017 Japanese Society of Allergology. Production and hosting by Elsevier

  20. Chemistry and biology of natural product derived protease inhibitors

    OpenAIRE

    Stolze, Sara Christina

    2012-01-01

    Im Rahmen dieser Dissertation sollten Naturstoffe und davon abgeleitete Derivate synthetisiert und im Hinblick auf ihre biologische Aktivität als Protease-Inhibitoren untersucht werden. Für die Naturstoffklasse der 3-Amino-6-Hydroxy-2-piperidon(Ahp)-Cyclodepsipeptide, die als nicht-kovalente Serin-Protease-Inhibitoren bekannt sind, konnte eine Festphasensynthese basierend auf einem allgemeinen Ahp-Vorläufermolekül entwickelt werden. Für den Ahp-Vorläufer wurde eine Lösungssynthese entwicke...

  1. A bacterial cysteine protease effector protein interferes with photosynthesis to suppress plant innate immune responses.

    Science.gov (United States)

    Rodríguez-Herva, José J; González-Melendi, Pablo; Cuartas-Lanza, Raquel; Antúnez-Lamas, María; Río-Alvarez, Isabel; Li, Ziduo; López-Torrejón, Gema; Díaz, Isabel; Del Pozo, Juan C; Chakravarthy, Suma; Collmer, Alan; Rodríguez-Palenzuela, Pablo; López-Solanilla, Emilia

    2012-05-01

    The bacterial pathogen Pseudomonas syringae pv tomato DC3000 suppresses plant innate immunity with effector proteins injected by a type III secretion system (T3SS). The cysteine protease effector HopN1, which reduces the ability of DC3000 to elicit programmed cell death in non-host tobacco, was found to also suppress the production of defence-associated reactive oxygen species (ROS) and callose when delivered by Pseudomonas fluorescens heterologously expressing a P. syringae T3SS. Purified His(6) -tagged HopN1 was used to identify tomato PsbQ, a member of the oxygen evolving complex of photosystem II (PSII), as an interacting protein. HopN1 localized to chloroplasts and both degraded PsbQ and inhibited PSII activity in chloroplast preparations, whereas a HopN1(D299A) non-catalytic mutant lost these abilities. Gene silencing of NtPsbQ in tobacco compromised ROS production and programmed cell death by DC3000. Our data reveal PsbQ as a contributor to plant immunity responses and a target for pathogen suppression. © 2012 Blackwell Publishing Ltd.

  2. Microbial electrosynthetic cells

    Energy Technology Data Exchange (ETDEWEB)

    May, Harold D.; Marshall, Christopher W.; Labelle, Edward V.

    2018-01-30

    Methods are provided for microbial electrosynthesis of H.sub.2 and organic compounds such as methane and acetate. Method of producing mature electrosynthetic microbial populations by continuous culture is also provided. Microbial populations produced in accordance with the embodiments as shown to efficiently synthesize H.sub.2, methane and acetate in the presence of CO.sub.2 and a voltage potential. The production of biodegradable and renewable plastics from electricity and carbon dioxide is also disclosed.

  3. Boosted protease inhibitors and the electrocardiographic measures of QT and PR durations

    DEFF Research Database (Denmark)

    Soliman, Elsayed Z; Lundgren, Jens D; Roediger, Mollie P

    2011-01-01

    There are contradictory reports regarding the effects of protease inhibitors on the ECG measures of QT and PR interval durations. The effect of interrupting use of protease inhibitors on QT and PR progression is also unknown.......There are contradictory reports regarding the effects of protease inhibitors on the ECG measures of QT and PR interval durations. The effect of interrupting use of protease inhibitors on QT and PR progression is also unknown....

  4. Semi-continuous in situ magnetic separation for enhanced extracellular protease productionmodeling and experimental validation

    DEFF Research Database (Denmark)

    Cerff, M.; Scholz, A.; Käppler, T.

    2013-01-01

    In modern biotechnology proteases play a major role as detergent ingredients. Especially the production of extracellular protease by Bacillus species facilitates downstream processing because the protease can be directly harvested from the biosuspension. In situ magnetic separation (ISMS...... production, and was used to optimize ISMS steps to obtain the maximum overall protease yield. Biotechnol. Bioeng. 2013; 110: 2161–2172. © 2013 Wiley Periodicals, Inc....

  5. Effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus.

    Science.gov (United States)

    Balasubramanian, Sivaraman A; Pye, David C; Willcox, Mark D P

    2013-03-01

    Proteases, protease activity and inflammatory molecules in tears have been found to be relevant in the pathogenesis of keratoconus. We sought to determine the influence of eye rubbing on protease expression, protease activity and concentration of inflammatory molecules in tears. Basal tears were collected from normal volunteers before and after 60 seconds of experimental eye rubbing. The total amount of matrix metalloproteinase (MMP)-13 and inflammatory molecules interleukin (IL)-6 and tumour necrosis factor (TNF)-α in the tear samples were measured using specific enzyme-linked immunosorbent assays (ELISA). Tear collagenase activity was investigated using a specific activity assay. The concentrations of MMP-13 (51.9 ± 34.3 versus 63 ± 36.8 pg/ml, p = 0.006), IL-6 (1.24 ± 0.98 versus 2.02 ± 1.52 pg/ml, p = 0.004) and TNF-α (1.16 ± 0.74 versus 1.44 ± 0.66 pg/ml, p = 0.003) were significantly increased in normal subjects after eye rubbing. The experimental eye rub did not alter significantly the collagenase activity (5.02 ± 3 versus 7.50 ± 3.90 fluorescent intensity units, p = 0.14) of tears. Eye rubbing for 60 seconds increased the level of tear MMP-13, IL-6 and TNF-α in normal study subjects. This increase in protease, protease activity and inflammatory mediators in tears after eye rubbing may be exacerbated even further during persistent and forceful eye rubbing seen in people with keratoconus and this in turn may contribute to the progression of the disease. © 2013 The Authors. Clinical and Experimental Optometry © 2013 Optometrists Association Australia.

  6. Characterization of the Aspergillus niger prtT, a unique regulator of extracellular protease encoding genes

    NARCIS (Netherlands)

    Punt, P.J.; Schuren, F.H.J.; Lehmbeck, J.; Christensen, T.; Hjort, C.; Hondel, C.A.M.J.J. van den

    2008-01-01

    Expression of several Aspergillus niger genes encoding major secreted, but not vacuolar, protease genes including the major acid protease gene pepA, was shown to be affected in the previously isolated A. niger protease mutant, AB1.13 [Mattern, I.E., van Noort, J.M., van den Berg, P., Archer, D.A.,

  7. Higher Desolvation Energy Reduces Molecular Recognition in Multi-Drug Resistant HIV-1 Protease

    Directory of Open Access Journals (Sweden)

    Ladislau C. Kovari

    2012-05-01

    Full Text Available Designing HIV-1 protease inhibitors that overcome drug-resistance is still a challenging task. In this study, four clinical isolates of multi-drug resistant HIV-1 proteases that exhibit resistance to all the US FDA-approved HIV-1 protease inhibitors and also reduce the substrate recognition ability were examined. A multi-drug resistant HIV-1 protease isolate, MDR 769, was co-crystallized with the p2/NC substrate and the mutated CA/p2 substrate, CA/p2 P1’F. Both substrates display different levels of molecular recognition by the wild-type and multi-drug resistant HIV-1 protease. From the crystal structures, only limited differences can be identified between the wild-type and multi-drug resistant protease. Therefore, a wild-type HIV-1 protease and four multi-drug resistant HIV-1 proteases in complex with the two peptides were modeled based on the crystal structures and examined during a 10 ns-molecular dynamics simulation. The simulation results reveal that the multi-drug resistant HIV-1 proteases require higher desolvation energy to form complexes with the peptides. This result suggests that the desolvation of the HIV-1 protease active site is an important step of protease-ligand complex formation as well as drug resistance. Therefore, desolvation energy could be considered as a parameter in the evaluation of future HIV-1 protease inhibitor candidates.

  8. Teaching Foundational Topics and Scientific Skills in Biochemistry within the Conceptual Framework of HIV Protease

    Science.gov (United States)

    Johnson, R. Jeremy

    2014-01-01

    HIV protease has served as a model protein for understanding protein structure, enzyme kinetics, structure-based drug design, and protein evolution. Inhibitors of HIV protease are also an essential part of effective HIV/AIDS treatment and have provided great societal benefits. The broad applications for HIV protease and its inhibitors make it a…

  9. 21 CFR 184.1027 - Mixed carbohydrase and protease enzyme product.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 3 2010-04-01 2009-04-01 true Mixed carbohydrase and protease enzyme product. 184... RECOGNIZED AS SAFE Listing of Specific Substances Affirmed as GRAS § 184.1027 Mixed carbohydrase and protease enzyme product. (a) Mixed carbohydrase and protease enzyme product is an enzyme preparation that includes...

  10. Identification and isoforms specificity of barley (Hordeum vulgare) grain proteinaceous inhibitors of commercial feed protease

    DEFF Research Database (Denmark)

    Dionisio, Giuseppe; Brinch-Pedersen, Henrik

    2016-01-01

    Protease is commonly used as feed additive. Ronozyme® ProAct, a subtilisin-like serine feed protease is different from the already characterized Bacillus subtilisin-like serine protease. When used in wheat and barley based feed, its degree of efficiency differs according to the cultivar in analys...

  11. A Kunitz-type cysteine protease inhibitor from cauliflower and Arabidopsis

    DEFF Research Database (Denmark)

    Halls, C.E.; Rogers, S. W.; Ouffattole, M.

    2006-01-01

    proaleurain maturation protease and of papain when assayed at pH 4.5 but not at pH 6.3. In a pull-down assay, the inhibitor bound tightly to papain, but only weakly to the aspartate protease pepsin. When the cauliflower protease inhibitor was transiently expressed in tobacco suspension culture protoplasts...

  12. Molecular Basis for Drug Resistance in HIV-1 Protease

    Directory of Open Access Journals (Sweden)

    Celia A. Schiffer

    2010-11-01

    Full Text Available HIV-1 protease is one of the major antiviral targets in the treatment of patients infected with HIV-1. The nine FDA approved HIV-1 protease inhibitors were developed with extensive use of structure-based drug design, thus the atomic details of how the inhibitors bind are well characterized. From this structural understanding the molecular basis for drug resistance in HIV-1 protease can be elucidated. Selected mutations in response to therapy and diversity between clades in HIV-1 protease have altered the shape of the active site, potentially altered the dynamics and even altered the sequence of the cleavage sites in the Gag polyprotein. All of these interdependent changes act in synergy to confer drug resistance while simultaneously maintaining the fitness of the virus. New strategies, such as incorporation of the substrate envelope constraint to design robust inhibitors that incorporate details of HIV-1 protease’s function and decrease the probability of drug resistance, are necessary to continue to effectively target this key protein in HIV-1 life cycle.

  13. Inactivation of proteinaceous protease inhibitors of soybeans by isolated fungi

    NARCIS (Netherlands)

    Meijer, M.M.T.; Spekking, W.T.J.; Sijtsma, L.; Bont, de J.A.M.

    1995-01-01

    Proteinaceous protease inhibitors, Kunitz Soybean Trypsin Inhibitor (KSTI) and Bowman Birk Inhibitor (BBI), in legume seeds reduce the digestibility of proteins in feed of monogastric animals. Enzymatic inactivation of these inhibitors will increase the nutritional value of the feed. The aim of this

  14. Breakdown of the innate immune system by bacterial proteases

    NARCIS (Netherlands)

    Laarman, A.J.

    2011-01-01

    Bacteria have developed many strategies to circumvent our immune system to survive and colonize human tissues. One of these strategies is by secreting proteases that specifically target the innate immune system. Aureolysin is a metalloprotease from Staphylococcus aureus which target the main

  15. Manipulating the autolytic pathway of a Bacillus protease

    NARCIS (Netherlands)

    VandenBurg, B; Eijsink, VGH; Vriend, G; Veltman, OR; Venema, G; HopsuHavu, VK; Jarvinen, M; Kirschke, H

    1997-01-01

    Autolytic degradation of Bacillus subtilis thermolysin-like proteinase (TLP-sub) is responsible for the irreversible inactivation of the enzyme at elevated temperatures. Previously, we reported five autolysis sites in B. subtilis neutral protease (Van den Burg et al., 1990, Biochem. J. 272:93-97).

  16. Alkaline protease production by alkaliphilic marine bacteria isolated ...

    African Journals Online (AJOL)

    The molecular mass determined using SDS-PAGE, was nearly 31.0 39 kDa. Some fundamental properties like effects of different temperatures, pH, metal ions (Ca2+, Mg2+, Cu2+, Pb3+, Mn2+ and Cd2+) and ethylene diamine tetraacetic acid (EDTA) on protease activity were also studied. Maximum activities were obtained ...

  17. Alkaline protease from senesced leaves of invasive weed Lantana ...

    African Journals Online (AJOL)

    STORAGESEVER

    2008-12-17

    Dec 17, 2008 ... amongst the most valuable commercial enzyme. Alkaline proteases hold a great potential for application in the detergent and leather industries (Kumar and Takagi,. 1999; Oberoi et al., 2001) due to the increasing trend to develop environmentally friendly technologies. Plants, animals and microbes are the ...

  18. Ionic liquids and proteases: A clean alliance for semisynthesis

    Czech Academy of Sciences Publication Activity Database

    Wehofsky, N.; Wespe, Ch.; Čeřovský, Václav; Pech, A.; Hoess, E.; Rudolph, R.; Bordusa, F.

    2008-01-01

    Roč. 9, č. 9 (2008), s. 1493-1499 ISSN 1439-4227 Grant - others:DFG(DE) SPP1191; DFG(DE) SFB610 Institutional research plan: CEZ:AV0Z40550506 Keywords : chemoenzymatic synthesis * ionic liquids * peptides * proteases * substrate mimetics Subject RIV: CC - Organic Chemistry Impact factor: 3.322, year: 2008

  19. Isolation of protease producing novel Bacillus cereus and detection ...

    African Journals Online (AJOL)

    user

    2011-02-14

    Feb 14, 2011 ... Key words: Protease, production, optimization, Bacillus sp. INTRODUCTION ... Nutrient broth (5 g peptone and 3 g meat extract, pH 7.0, Merck) was used as the common growth ... nitrate through nitrite. It was determined that ...

  20. Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

    Czech Academy of Sciences Publication Activity Database

    Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

    2015-01-01

    Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

  1. An oxidant, detergent and salt stable alkaline protease from Bacillus ...

    African Journals Online (AJOL)

    A novel soil bacterium, Bacillus cereus SIU1 was earlier isolated from non-saline, slightly alkaline soil of Eastern Uttar Pradesh, India. The isolate B. cereus SIU1 was grown in modified glucose yeast extract (modified GYE) medium at pH 9.0 and 45°C. It produced maximum protease at 20 h incubation. The enzyme was ...

  2. Milk Clotting Activity of Protease, Extracted from Rhizome of Taffin ...

    African Journals Online (AJOL)

    MBI

    2017-03-07

    Mar 7, 2017 ... The increasing prices of calf rennets, their accessibility and ethical concerns ... the region with a massive annual production (FAO, ... valuable group of enzymes with various industrial ... use of protease enzymes in the food industry .... In the procedure, Bovine Serum Albumin ..... Agricultural Economics.

  3. Purification and characterization of protease from Bacillus cereus ...

    African Journals Online (AJOL)

    chitti

    2013-09-16

    Sep 16, 2013 ... Purification and characterization of protease from. Bacillus cereus SU12 isolated from oyster. Saccostrea cucullata. S. Umayaparvathi*, S. Meenakshi, M. Arumugam and T. Balasubramanian. Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai - 608.

  4. Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication.

    Science.gov (United States)

    Song, Qin-Qin; Lu, Ming-Zhi; Song, Juan; Chi, Miao-Miao; Sheng, Lin-Jun; Yu, Jie; Luo, Xiao-Nuan; Zhang, Lu; Yao, Hai-Lan; Han, Jun

    2015-10-02

    To determine whether 2A protease of the enterovirus genus with type I internal ribosome entry site (IRES) effect on the viral replication of type II IRES, coxsackievirus B3(CVB3)-encoded protease 2A and encephalomyocarditis virus (EMCV) IRES (Type II)-dependent or cap-dependent report gene were transiently co-expressed in eukaryotic cells. We found that CVB3 2A protease not only inhibited translation of cap-dependent reporter genes through the cleavage of eIF4GI, but also conferred high EMCV IRES-dependent translation ability and promoted EMCV replication. Moreover, deletions of short motif (aa13-18 RVVNRH, aa65-70 KNKHYP, or aa88-93 PRRYQSH) resembling the nuclear localization signals (NLS) or COOH-terminal acidic amino acid motif (aa133-147 DIRDLLWLEDDAMEQ) of CVB3 2A protease decreased both its EMCV IRES-dependent translation efficiency and destroy its cleavage on eukaryotic initiation factor 4G (eIF4G) I. Our results may provide better understanding into more effective interventions and treatments for co-infection of viral diseases. Copyright © 2015 Elsevier B.V. All rights reserved.

  5. Targeted degradomics in protein terminomics and protease substrate discovery

    DEFF Research Database (Denmark)

    Savickas, Simonas; auf dem Keller, Ulrich

    2017-01-01

    extensive degradomics target lists that now can be tested with help of selected and parallel reaction monitoring (S/PRM) in complex biological systems, where proteases act in physiological environments. In this minireview, we describe the general principles of targeted degradomics, outline the generic...

  6. The non-death role of metacaspase proteases

    International Nuclear Information System (INIS)

    Shrestha, Amit; Megeney, Lynn A.

    2012-01-01

    The activation of caspase proteases and the targeting of protein substrates act as key steps in the engagement and conduct of apoptosis/programmed cell death. However, the discovery of caspase involvement in diverse non-apoptotic cellular functions strongly suggests that these proteins may have evolved from a core behavior unrelated to the induction of cell death. The presence of similar proteases, termed metacaspases, in single cell organisms supports the contention that such proteins may have co-evolved or derived from a critical non-death function. Indeed, the benefit(s) for single cell life forms to retain proteins solely dedicated to self destruction would be countered by a strong selection pressure to curb or eliminate such processes. Examination of metacaspase biology provides evidence that these ancient protease forerunners of the caspase family also retain versatility in function, i.e., death and non-death cell functions. Here, we provide a critical review that highlights the non-death roles of metacaspases that have been described thus far, and the impact that these observations have for our understanding of the evolution and cellular utility of this protease family.

  7. Purification and characterization of a protease from Thermophilic ...

    African Journals Online (AJOL)

    AJB SERVER

    2006-10-19

    Oct 19, 2006 ... protein liquid chromatography. The method gave a ... gent industry are the proteases from bacteria sources ... In this paper, we report our recent progress on the purification ... 10 to 60 min, then cooled in ice-water and the residue activity was measured .... Huo P, Mao J, Shi Y (2003). ... Kumar CG (2002).

  8. Retroviral proteases and their roles in virion maturation

    Czech Academy of Sciences Publication Activity Database

    Konvalinka, Jan; Kräusslich, H. G.; Müller, B.

    2015-01-01

    Roč. 479, SI (2015), s. 403-417 ISSN 0042-6822 R&D Projects: GA ČR GBP208/12/G016; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : retrovirus * aspartic protease * maturation * human immunodeficiency virus * Gag Subject RIV: CE - Biochemistry Impact factor: 3.200, year: 2015

  9. Physical and chemical properties of the acid protease from ...

    African Journals Online (AJOL)

    samsung

    2016-03-02

    Mar 2, 2016 ... is the principal set of biochemical changes during ... coagulating ability by analysis of the products of casein ... for protease activity, milk-clotting activity and protein content. ..... Figure 5, the content of casein components decreased in .... Purification, caracterization, molecular cloning and modelling of its.

  10. Extracellular acid protease from Aspergillus niger I1: purification and ...

    African Journals Online (AJOL)

    STORAGESEVER

    2009-09-15

    Sep 15, 2009 ... A new strain of Aspergillus niger producing acid protease was isolated and identified by universal primers NL1 and .... Media were autoclaved at 120°C for 20 min. ... molecular weight calibration kit as markers consisting of bovine ... then removed by washing the gel three times with 100 mM ..... New York.

  11. Serine protease from midgut of Bombus terrestris males

    Czech Academy of Sciences Publication Activity Database

    Brabcová, Jana; Kindl, Jiří; Valterová, Irena; Pichová, Iva; Zarevúcka, Marie; Brabcová, J.; Jágr, Michal; Mikšík, Ivan

    2013-01-01

    Roč. 82, č. 3 (2013), s. 117-128 ISSN 0739-4462 R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Bombus terrestris * midgut * serine protease * bumblebee Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 1.160, year: 2013

  12. Optimization of protease production by an actinomycete Strain, PS ...

    African Journals Online (AJOL)

    STORAGESEVER

    distilled water and this was inoculated into 5 ml of gelatin broth and .... leakage. After that, the dialysis bag was suspended in a beaker containing 0.5 M Tris-HCL buffer (pH 8.5) for 24 h, ... the detection of optimum temperature for the protease.

  13. 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...

  14. Production of alkaline protease by Teredinobacter turnirae cells ...

    African Journals Online (AJOL)

    The conditions for immobilizing the new alkaline protease-producing bacteria strain Teredinobacter turnirae by entrapment in calcium alginate gel were investigated. The influence of alginate concentration (20, 25 and 30 g/l) and initial cell loading (ICL) on enzyme production were studied. The production of alkaline ...

  15. Delay of Iris flower senescence by protease inhibitors

    NARCIS (Netherlands)

    Pak, C.; Doorn, van W.G.

    2005-01-01

    asterisk inside a circle sign Visible senescence of the flag tepals in Iris x hollandica (cv. Blue Magic) was preceded by a large increase in endoprotease activity. Just before visible senescence about half of total endoprotease activity was apparently due to cysteine proteases, somewhat less than

  16. molecular biology approach to the search for novel hiv proteases ...

    African Journals Online (AJOL)

    ... which could be tested in the animal models of HIV infection before subjection to clinical trials. Optimistically, the magic HIV therapeutics may be hidden in such insects and may require the application of molecular biology techniques to unravel. KEY WORDS: Antiretroviral drugs, malaria, proteases, restriction enzymes, ...

  17. Genome-reconstruction for eukaryotes from complex natural microbial communities.

    Science.gov (United States)

    West, Patrick T; Probst, Alexander J; Grigoriev, Igor V; Thomas, Brian C; Banfield, Jillian F

    2018-04-01

    Microbial eukaryotes are integral components of natural microbial communities, and their inclusion is critical for many ecosystem studies, yet the majority of published metagenome analyses ignore eukaryotes. In order to include eukaryotes in environmental studies, we propose a method to recover eukaryotic genomes from complex metagenomic samples. A key step for genome recovery is separation of eukaryotic and prokaryotic fragments. We developed a k -mer-based strategy, EukRep, for eukaryotic sequence identification and applied it to environmental samples to show that it enables genome recovery, genome completeness evaluation, and prediction of metabolic potential. We used this approach to test the effect of addition of organic carbon on a geyser-associated microbial community and detected a substantial change of the community metabolism, with selection against almost all candidate phyla bacteria and archaea and for eukaryotes. Near complete genomes were reconstructed for three fungi placed within the Eurotiomycetes and an arthropod. While carbon fixation and sulfur oxidation were important functions in the geyser community prior to carbon addition, the organic carbon-impacted community showed enrichment for secreted proteases, secreted lipases, cellulose targeting CAZymes, and methanol oxidation. We demonstrate the broader utility of EukRep by reconstructing and evaluating relatively high-quality fungal, protist, and rotifer genomes from complex environmental samples. This approach opens the way for cultivation-independent analyses of whole microbial communities. © 2018 West et al.; Published by Cold Spring Harbor Laboratory Press.

  18. In-cell protease assay systems based on trans-localizing molecular beacon proteins using HCV protease as a model system.

    Directory of Open Access Journals (Sweden)

    Jeong Hee Kim

    Full Text Available This study describes a sensitive in-cell protease detection system that enables direct fluorescence detection of a target protease and its inhibition inside living cells. This live-cell imaging system provides a fluorescent molecular beacon protein comprised of an intracellular translocation signal sequence, a protease-specific cleavage sequence, and a fluorescent tag sequence(s. The molecular beacon protein is designed to change its intracellular localization upon cleavage by a target protease, i.e., from the cytosol to a subcellular organelle or from a subcellular organelle to the cytosol. Protease activity can be monitored at the single cell level, and accordingly the entire cell population expressing the protease can be accurately enumerated. The clear cellular change in fluorescence pattern makes this system an ideal tool for various life science and drug discovery research, including high throughput and high content screening applications.

  19. Microbial accumulation of uranium

    International Nuclear Information System (INIS)

    Zhang Wei; Dong Faqin; Dai Qunwei

    2005-01-01

    The mechanism of microbial accumulation of uranium and the effects of some factors (including pH, initial uranium concentration, pretreatment of bacteria, and so on) on microbial accumulation of uranium are discussed briefly. The research direction and application prospect are presented. (authors)

  20. MICROBIAL FUEL CELL

    DEFF Research Database (Denmark)

    2008-01-01

    A novel microbial fuel cell construction for the generation of electrical energy. The microbial fuel cell comprises: (i) an anode electrode, (ii) a cathode chamber, said cathode chamber comprising an in let through which an influent enters the cathode chamber, an outlet through which an effluent...

  1. Microbial control of pollution

    Energy Technology Data Exchange (ETDEWEB)

    Fry, J C; Gadd, G M; Herbert, R A; Jones, C W; Watson-Craik, I A [eds.

    1992-01-01

    12 papers are presented on the microbial control of pollution. Topics covered include: bioremediation of oil spills; microbial control of heavy metal pollution; pollution control using microorganisms and magnetic separation; degradation of cyanide and nitriles; nitrogen removal from water and waste; and land reclamation and restoration.

  2. Identification, Characterization and Down-Regulation of Cysteine Protease Genes in Tobacco for Use in Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Kishor Duwadi

    Full Text Available Plants are an attractive host system for pharmaceutical protein production. Many therapeutic proteins have been produced and scaled up in plants at a low cost compared to the conventional microbial and animal-based systems. The main technical challenge during this process is to produce sufficient levels of recombinant proteins in plants. Low yield is generally caused by proteolytic degradation during expression and downstream processing of recombinant proteins. The yield of human therapeutic interleukin (IL-10 produced in transgenic tobacco leaves was found to be below the critical level, and may be due to degradation by tobacco proteases. Here, we identified a total of 60 putative cysteine protease genes (CysP in tobacco. Based on their predicted expression in leaf tissue, 10 candidate CysPs (CysP1-CysP10 were selected for further characterization. The effect of CysP gene silencing on IL-10 accumulation was examined in tobacco. It was found that the recombinant protein yield in tobacco could be increased by silencing CysP6. Transient expression of CysP6 silencing construct also showed an increase in IL-10 accumulation in comparison to the control. Moreover, CysP6 localizes to the endoplasmic reticulum (ER, suggesting that ER may be the site of IL-10 degradation. Overall results suggest that CysP6 is important in determining the yield of recombinant IL-10 in tobacco leaves.

  3. Interdependence of Inhibitor Recognition in HIV-1 Protease.

    Science.gov (United States)

    Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

    2017-05-09

    Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.

  4. Effects of cysteine protease inhibitors on rabbit cathepsin D maturation

    International Nuclear Information System (INIS)

    Samarel, A.M.; Ferguson, A.G.; Decker, R.S.; Lesch, M.

    1989-01-01

    To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation

  5. HIV protease inhibitors in pregnancy : pharmacology and clinical use.

    Science.gov (United States)

    Andany, Nisha; Loutfy, Mona R

    2013-03-01

    The impact of antiretroviral therapy (ART) on the natural history of HIV-1 infection has resulted in dramatic reductions in disease-associated morbidity and mortality. Additionally, the epidemiology of HIV-1 infection worldwide is changing, as women now represent a substantial proportion of infected adults. As more highly effective and tolerable antiretroviral regimens become available, and as the prevention of mother-to-child transmission becomes an attainable goal in the management of HIV-infected individuals, more and more HIV-positive women are choosing to become pregnant and have children. Consequently, it is important to consider the efficacy and safety of antiretroviral agents in pregnancy. Protease inhibitors are a common class of medication used in the treatment of HIV-1 infection and are increasingly being used in pregnancy. However, several studies have raised concerns regarding pharmacokinetic alterations in pregnancy, particularly in the third trimester, which results in suboptimal drug concentrations and a theoretically higher risk of virologic failure and perinatal transmission. Drug level reductions have been observed with each individual protease inhibitor and dose adjustments in pregnancy are suggested for certain agents. Furthermore, studies have also raised concerns regarding the safety of protease inhibitors in pregnancy, particularly as they may increase the risk of pre-term birth and metabolic disturbances. Overall, protease inhibitors are safe and effective for the treatment of HIV-infected pregnant women. Specifically, ritonavir-boosted lopinavir- and atazanavir-based regimens are preferred in pregnancy, while ritonavir-boosted darunavir- and saquinavir-based therapies are reasonable alternatives. This paper reviews the use of protease inhibitors in pregnancy, focusing on pharmacokinetic and safety considerations, and outlines the recommendations for use of this class of medication in the HIV-1-infected pregnant woman.

  6. 10 CFR 15.41 - When a claim may be compromised.

    Science.gov (United States)

    2010-01-01

    ... it has not been referred to DOJ for litigation. (b) Unless otherwise provided by law, when the... with the DOJ. The NRC will evaluate the compromise offer, using the factors set forth in this part. If an offer to compromise any debt in excess of $100,000 is acceptable to the NRC, the NRC shall refer...

  7. 32 CFR 842.99 - Compromise, termination, and suspension of collection.

    Science.gov (United States)

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Compromise, termination, and suspension of collection. 842.99 Section 842.99 National Defense Department of Defense (Continued) DEPARTMENT OF THE AIR... States (31 U.S.C. 3701, 3711-3719) § 842.99 Compromise, termination, and suspension of collection. This...

  8. Near-infrared spectroscopy for detection of vascular compromise in paediatric supracondylar fractures

    International Nuclear Information System (INIS)

    Skowno, Justin J; De Lima, Jonathan; Quick, Tom J; Carpenter, Eleanor C; Gibbons, Paul J; Little, David G

    2014-01-01

    Children suffering supracondylar fractures of the humerus are at risk of vascular compromise, which is currently assessed clinically, although other modalities such as angiography, pulse oximetry, Doppler ultrasound and magnetic resonance angiography have been used. We sought to ascertain whether tissue haemoglobin oxygenation (StO 2 ) measurement could distinguish between patients with and without clinical vascular compromise following supracondylar fractures of the humerus. We prospectively observed StO 2  using near-infrared spectroscopy in 29 paediatric patients with supracondylar fractures requiring operative manipulation. The injured and uninjured volar forearm compartments were monitored immediately before and after fracture reduction. The relationship between StO 2  in the injured and uninjured limb, and the presence of pre-operative vascular compromise was assessed. Seven out of 29 children presented with vascular compromise. Patients with clinical vascular compromise had significantly lower pre-reduction StO 2  (63.5% ± 15%, mean ± standard deviation), compared to those without compromise (80.9% ± 10%). StO 2  normalized following surgery in all children with vascular compromise. These improvements in muscle StO 2  were associated, in all patients, with the clinical return of pulses and resolution of neurological symptoms if present. StO 2  monitoring can identify patients with clinical vascular compromise, can identify the return of adequate perfusion following operative correction of supracondylar fractures, and may be a useful adjunct to clinical assessment. (paper)

  9. 7 CFR 1956.68 - Compromise or adjustment without debtor's signature.

    Science.gov (United States)

    2010-01-01

    ... 7 Agriculture 14 2010-01-01 2009-01-01 true Compromise or adjustment without debtor's signature... Loan Programs and Multi-Family Housing § 1956.68 Compromise or adjustment without debtor's signature... made to obtain the debtor's signature and the date(s) of such effort. (c) The specific reasons why it...

  10. 36 CFR 1011.7 - When will the Presidio Trust compromise a debt?

    Science.gov (United States)

    2010-07-01

    ... 36 Parks, Forests, and Public Property 3 2010-07-01 2010-07-01 false When will the Presidio Trust... Procedures To Collect Presidio Trust Debts § 1011.7 When will the Presidio Trust compromise a debt? (a... debt owed to the Presidio Trust that is not recovered as the result of a compromise will be reported to...

  11. 26 CFR 301.6331-3 - Restrictions on levy while offers to compromise are pending.

    Science.gov (United States)

    2010-04-01

    ... are pending. 301.6331-3 Section 301.6331-3 Internal Revenue INTERNAL REVENUE SERVICE, DEPARTMENT OF... for Collection of Taxes § 301.6331-3 Restrictions on levy while offers to compromise are pending. Cross-reference. For provisions relating to the making of levies while an offer to compromise is pending...

  12. 15 CFR 19.7 - When will Commerce entities compromise a Commerce debt?

    Science.gov (United States)

    2010-01-01

    ... 15 Commerce and Foreign Trade 1 2010-01-01 2010-01-01 false When will Commerce entities compromise a Commerce debt? 19.7 Section 19.7 Commerce and Foreign Trade Office of the Secretary of Commerce COMMERCE DEBT COLLECTION Procedures To Collect Commerce Debts § 19.7 When will Commerce entities compromise...

  13. A role in immunity for Arabidopsis cysteine protease RD21, the ortholog of the tomato immune protease C14.

    Directory of Open Access Journals (Sweden)

    Takayuki Shindo

    Full Text Available Secreted papain-like Cys proteases are important players in plant immunity. We previously reported that the C14 protease of tomato is targeted by cystatin-like EPIC proteins that are secreted by the oomycete pathogen Phytophthora infestans (Pinf during infection. C14 has been under diversifying selection in wild potato species coevolving with Pinf and reduced C14 levels result in enhanced susceptibility for Pinf. Here, we investigated the role C14-EPIC-like interactions in the natural pathosystem of Arabidopsis with the oomycete pathogen Hyaloperonospora arabidopsidis (Hpa. In contrast to the Pinf-solanaceae pathosystem, the C14 orthologous protease of Arabidopsis, RD21, does not evolve under diversifying selection in Arabidopsis, and rd21 null mutants do not show phenotypes upon compatible and incompatible Hpa interactions, despite the evident lack of a major leaf protease. Hpa isolates express highly conserved EPIC-like proteins during infections, but it is unknown if these HpaEPICs can inhibit RD21 and one of these HpaEPICs even lacks the canonical cystatin motifs. The rd21 mutants are unaffected in compatible and incompatible interactions with Pseudomonas syringae pv. tomato, but are significantly more susceptible for the necrotrophic fungal pathogen Botrytis cinerea, demonstrating that RD21 provides immunity to a necrotrophic pathogen.

  14. Some Investigations on Protease Enzyme Production Kinetics Using Bacillus licheniformis BBRC 100053 and Effects of Inhibitors on Protease Activity

    Directory of Open Access Journals (Sweden)

    Zahra Ghobadi Nejad

    2014-01-01

    Full Text Available Due to great commercial application of protease, it is necessary to study kinetic characterization of this enzyme in order to improve design of enzymatic reactors. In this study, mathematical modeling of protease enzyme production kinetics which is derived from Bacillus licheniformis BBRC 100053 was studied (at 37°C, pH 10 after 73 h in stationary phase, and 150 rpm. The aim of the present paper was to determine the best kinetic model and kinetic parameters for production of protease and calculating Ki (inhibition constant of different inhibitors to find the most effective one. The kinetic parameters Km (Michaelis-Menten constant and Vm (maximum rate were calculated 0.626 mM and 0.0523 mM/min. According to the experimental results, using DFP (diisopropyl fluorophosphate and PMSF (phenylmethanesulfonyl fluoride as inhibitors almost 50% of the enzyme activity could be inhibited when their concentrations were 0.525 and 0.541 mM, respectively. Ki for DFP and PMSF were 0.46 and 0.56 mM, respectively. Kinetic analysis showed that the Lineweaver-Burk model was the best fitting model for protease production kinetics DFP was more effective than PMSF and both of them should be covered in the group of noncompetitive inhibitors.

  15. Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

    Science.gov (United States)

    Valls, Cristina; Pujadas, Gerard; Garcia-Vallve, Santi; Mulero, Miquel

    2011-01-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

  16. Identification of cysteine proteases and screening of cysteine protease inhibitors in biological samples by a two-dimensional gel system of zymography and reverse zymography.

    Science.gov (United States)

    Saitoh, Eiichi; Yamamoto, Shinya; Okamoto, Eishiro; Hayakawa, Yoshimi; Hoshino, Takashi; Sato, Ritsuko; Isemura, Satoko; Ohtsubo, Sadami; Taniguchi, Masayuki

    2007-11-18

    We have developed a two-dimensional (2D-) gel system of zymography and reverse zymography for the detection and characterization of proteases and protease inhibitors. Isoelectric focusing (IEF) agarose gels with pH gradients were employed for separation in the first-dimension and sodium dodecyl sulfate (SDS)-polyacrylamide gel copolymerized with gelatin used for the second dimension. Proteases and protease inhibitors separated by IEF gel were applied on the second gel without trichloroacetic acid (TCA) fixation. Protease activity in the 2D-gel was visualized as transparent spots where gelatin substrate was digested after commassie brilliant blue (CBB) staining. Some of the transparent spots from the skin mucus extract of rainbow trout were determined to be a cysteine protease through use of E-64 or CA-074. In the reverse zymography technique, the gel was incubated with papain solution at 37 degrees C for 18 h. Cysteine protease inhibitors from broad bean seeds were detected as clear blue spots after CBB staining. The amino (N-) terminal sequences of four papain inhibitor spots thus detected were demonstrated to be identical to that of favin beta chain, a broad bean lectin. Taken together, our system can be considered to be an efficient technique for discovering and characterizing new proteases and protease inhibitors in biological samples. This is the first report describing a 2D-gel system of zymography and reverse zymography.

  17. The waaL gene mutation compromised the inhabitation of Enterobacter sp. Ag1 in the mosquito gut environment.

    Science.gov (United States)

    Pei, Dong; Jiang, Jinjin; Yu, Wanqin; Kukutla, Phanidhar; Uentillie, Alejandro; Xu, Jiannong

    2015-08-27

    The mosquito gut harbors a variety of bacteria that are dynamically associated with mosquitoes in various contexts. However, little is known about bacterial factors that affect bacterial inhabitation in the gut microbial community. Enterobacter sp. Ag1 is a predominant Gram negative bacterium in the mosquito midgut. In a mutant library that was generated using transposon Tn5-mediated mutagenesis, a mutant was identified, in which the gene waaL was disrupted by the Tn5 insertion. The waaL encodes O antigen ligase, which is required for the attachment of O antigen to the outer core oligosaccharide of the lipopolysaccharide (LPS). The waaL(-) mutation caused the O antigen repeat missing in the LPS. The normal LPS structure was restored when the mutant was complemented with a plasmid containing waaL gene. The waaL(-) mutation did not affect bacterial proliferation in LB culture, the mutant cells grew at a rate the same as the wildtype (wt) cells. However, when waaL(-) strain were co-cultured with the wt strain or complemented strain, the mutant cells proliferated with a slower rate, indicating that the mutants were less competitive than wt cells in a community setting. Similarly, in a co-feeding assay, when fluorescently tagged wt strain and waaL(-) strain were orally co-introduced into the gut of Anopheles stephensi mosquitoes, the mutant cells were less prevalent in both sugar-fed and blood-fed guts. The data suggest that the mutation compromised the bacterial inhabitation in the gut community. Besides, the mutant was more sensitive to oxidative stress, demonstrated by lower survival rate upon exposure to 20 mM H₂O₂. Lack of the O antigen structure in LPS of Enterobacter compromised the effective growth in co-culture and co-feeding assays. In addition, O-antigen was involved in protection against oxidative stress. The findings suggest that intact LPS is crucial for the bacteria to steadily stay in the gut microbial community.

  18. Chimeric exchange of coronavirus nsp5 proteases (3CLpro) identifies common and divergent regulatory determinants of protease activity.

    Science.gov (United States)

    Stobart, Christopher C; Sexton, Nicole R; Munjal, Havisha; Lu, Xiaotao; Molland, Katrina L; Tomar, Sakshi; Mesecar, Andrew D; Denison, Mark R

    2013-12-01

    Human coronaviruses (CoVs) such as severe acute respiratory syndrome CoV (SARS-CoV) and Middle East respiratory syndrome CoV (MERS-CoV) cause epidemics of severe human respiratory disease. A conserved step of CoV replication is the translation and processing of replicase polyproteins containing 16 nonstructural protein domains (nsp's 1 to 16). The CoV nsp5 protease (3CLpro; Mpro) processes nsp's at 11 cleavage sites and is essential for virus replication. CoV nsp5 has a conserved 3-domain structure and catalytic residues. However, the intra- and intermolecular determinants of nsp5 activity and their conservation across divergent CoVs are unknown, in part due to challenges in cultivating many human and zoonotic CoVs. To test for conservation of nsp5 structure-function determinants, we engineered chimeric betacoronavirus murine hepatitis virus (MHV) genomes encoding nsp5 proteases of human and bat alphacoronaviruses and betacoronaviruses. Exchange of nsp5 proteases from HCoV-HKU1 and HCoV-OC43, which share the same genogroup, genogroup 2a, with MHV, allowed for immediate viral recovery with efficient replication albeit with impaired fitness in direct competition with wild-type MHV. Introduction of MHV nsp5 temperature-sensitive mutations into chimeric HKU1 and OC43 nsp5 proteases resulted in clear differences in viability and temperature-sensitive phenotypes compared with MHV nsp5. These data indicate tight genetic linkage and coevolution between nsp5 protease and the genomic background and identify differences in intramolecular networks regulating nsp5 function. Our results also provide evidence that chimeric viruses within coronavirus genogroups can be used to test nsp5 determinants of function and inhibition in common isogenic backgrounds and cell types.

  19. The threonine protease activity of testes-specific protease 50 (TSP50 is essential for its function in cell proliferation.

    Directory of Open Access Journals (Sweden)

    Yu-Yin Li

    Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50, a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.

  20. Proteases in Escherichia coli and Staphylococcus aureus confer reduced susceptibility to lactoferricin B.

    Science.gov (United States)

    Ulvatne, Hilde; Haukland, Hanne Husom; Samuelsen, Ørjan; Krämer, Manuela; Vorland, Lars H

    2002-10-01

    Lactoferricin B is a cationic antimicrobial peptide derived from the N-terminal part of bovine lactoferrin. The effect of bacterial proteases on the antibacterial activity of lactoferricin B towards Escherichia coli and Staphylococcus aureus was investigated using various protease inhibitors and protease-deficient E. coli mutants. Sodium-EDTA, a metalloprotease inhibitor, was the most efficient inhibitors in both species, but combinations of sodium-EDTA with other types of protease inhibitor gave a synergic effect. The results indicate that several groups of proteases are involved in resistance to lactoferricin B in both E. coli and S. aureus. We also report that genetic inactivation of the heat shock-induced serine protease DegP increased the susceptibility to lactoferricin B in E. coli, suggesting that this protease, at least, is involved in reduced susceptibility to lactoferricin B.

  1. Production and partial characterization of alkaline protease from bacillus subtilis mutant induced by gamma radiation

    International Nuclear Information System (INIS)

    Ibrahim, H.M.M.; Bashandy, A.S.

    2010-01-01

    Fourteen bacterial isolates belonging to B.subtilis were locally isolated from soil and screened for alkaline protease production. Only one strain, the highly potent one, was selected as alkaline protease producer and subjected to further studies to optimize its production. Alkaline protease production was maximum at 35 degree C after 72 h of incubation and at ph 10.0. molasses as a carbon source and combination of peptone and yeast extract as a nitrogen source enhanced greatly alkaline protease production. The mutant strain induced by gamma radiation showed higher alkaline protease production by 1.97 fold as compared with the parent strain. The alkaline protease enzyme was active at 40 degree C and ph 10. It was compatible with many commercial detergents and showed high stability (84 %) of its original activity with Ariel detergent. Moreover, alkaline protease enhanced the washing performance, and retained 95 % of its activity in the formulated dry powder.

  2. A signaling protease required for melanization in Drosophila affects resistance and tolerance of infections.

    Directory of Open Access Journals (Sweden)

    Janelle S Ayres

    2008-12-01

    Full Text Available Organisms evolve two routes to surviving infections-they can resist pathogen growth (resistance and they can endure the pathogenesis of infection (tolerance. The sum of these two properties together defines the defensive capabilities of the host. Typically, studies of animal defenses focus on either understanding resistance or, to a lesser extent, tolerance mechanisms, thus providing little understanding of the relationship between these two mechanisms. We suggest there are nine possible pairwise permutations of these traits, assuming they can increase, decrease, or remain unchanged in an independent manner. Here we show that by making a single mutation in the gene encoding a protease, CG3066, active in the melanization cascade in Drosophila melanogaster, we observe the full spectrum of changes; these mutant flies show increases and decreases in their resistance and tolerance properties when challenged with a variety of pathogens. This result implicates melanization in fighting microbial infections and shows that an immune response can affect both resistance and tolerance to infections in microbe-dependent ways. The fly is often described as having an unsophisticated and stereotypical immune response where single mutations cause simple binary changes in immunity. We report a level of complexity in the fly's immune response that has strong ecological implications. We suggest that immune responses are highly tuned by evolution, since selection for defenses that alter resistance against one pathogen may change both resistance and tolerance to other pathogens.

  3. A Novel Trp-rich Model Antimicrobial Peptoid with Increased Protease Stability

    International Nuclear Information System (INIS)

    Bang, Jeong Kyu; Nan, Yong Hai; Lee, Eun Kyu; Shin, Song Yub

    2010-01-01

    In order to increase protease stability of a novel Trp-rich model antimicrobial peptide, K 6 L 2 W 3 (KLWKKWKKWLKNH 2 ) and investigate the effect of L-amino acid to peptoid residue conversion on biological functions, we synthesized its antimicrobial peptoid, k 6 l 2 w 3 . Peptoid k 6 l 2 w 3 had similar bacterial selectivity compared to peptide K 6 L 2 W 3 . The bactericidal rate of k 6 l 2 w 3 was somewhat slower than that of K 6 L 2 W 3 . Peptoid k 6 l 2 w 3 exhibited very little dye leakage from bacterial outer-membrane mimicking PE/PG liposomes, as observed in K 6 L 2 W 3 , indicating that the major target site of K 6 L 2 W 3 and k 6 l 2 w 3 may be not the cell membrane but the cytoplasm of bacteria. Trypsin treatment of K 6 L 2 W 3 completely abolished antimicrobial activities against Escherichia coli and Staphylococcus aureus. In contrast, the antimicrobial activity of k 6 l 2 w 3 was completely preserved after trypsin treatment. Taken together, our results suggested that antimicrobial peptoid k 6 l 2 w 3 can potentially serves as a promising therapeutic agent for the treatment of microbial infection

  4. Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

    Science.gov (United States)

    Maddur, Ashoka A; Swanson, Richard; Izaguirre, Gonzalo; Gettins, Peter G W; Olson, Steven T

    2013-11-01

    Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.

  5. A low-cost, multiplexable, automated flow cytometry procedure for the characterization of microbial stress dynamics in bioreactors

    DEFF Research Database (Denmark)

    Brognaux, Alison; Han, Shanshan; Sørensen, Søren Johannes

    2013-01-01

    BACKGROUND:Microbial cell population heterogeneity is now recognized as a major source of issues in the development and optimization of bioprocesses. Even if single cell technologies are available for the study of microbial population heterogeneity, only a few of these methods are available...... to the protease content and energy status of the cells. In this respect, multiplexed experiments have shown a correlation between heat shock and ATP content and the stability of the reporter molecule.CONCLUSION:This work demonstrates that a simplified version of on-line FC can be used at the process level...

  6. Cardiovascular disease and use of contemporary protease inhibitors

    DEFF Research Database (Denmark)

    Ryom, Lene; Lundgren, Jens D; El-Sadr, Wafaa

    2018-01-01

    BACKGROUND: Although earlier protease inhibitors have been associated with increased risk of cardiovascular disease, whether this increased risk also applies to more contemporary protease inhibitors is unknown. We aimed to assess whether cumulative use of ritonavir-boosted atazanavir and ritonavir......-boosted darunavir were associated with increased incidence of cardiovascular disease in people living with HIV. METHODS: The prospective Data Collection on Adverse Events of Anti-HIV Drugs (D:A:D) study consists of people living with HIV-1 from 11 cohorts in Australia, Europe, and the USA. Participants were...... monitored from Jan 1, 2009, until the earliest of a cardiovascular event, 6 months after the last visit, or until Feb 1, 2016. The outcome of interest was the incidence of cardiovascular disease in adults (aged ≥16 years) living with HIV who were being treated with contemporary treatments. We defined...

  7. Advances in zymography techniques and patents regarding protease analysis.

    Science.gov (United States)

    Wilkesman, Jeff; Kurz, Liliana

    2012-08-01

    Detection of enzymatic activity on gel electrophoresis, namely zymography, is a technique that has received increasing attention in the last 10 years, according to the number of articles published. A growing amount of enzymes, mainly proteases, are now routinely detected by zymography. Detailed analytical studies are beginning to be published, as well as new patents have been developed. This new article updates the information covered in our last review, condensing the recent publications dealing with the identification of proteolytic enzymes in electrophoretic gel supports and its variations. The new advances of this method are basically focused towards two dimensional zymography and transfer zymography. Though comparatively fewer patents have been published, they basically coincide in the study of matrix metalloproteases. The tendency is foreseen to be very productive in the area of zymoproteomics, combining electrophoresis and mass spectrometry for the analysis of proteases.

  8. Effects of Mucuna pruriens protease inhibitors on Echis carinatus venom.

    Science.gov (United States)

    Hope-Onyekwere, Nnadozie Stanley; Ogueli, Godwin Ifeanyi; Cortelazzo, Alessio; Cerutti, Helena; Cito, Annarita; Aguiyi, John C; Guerranti, Roberto

    2012-12-01

    The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay. Copyright © 2012 John Wiley & Sons, Ltd.

  9. Intervention with Serine Protease Activity with Small Peptides

    DEFF Research Database (Denmark)

    Xu, Peng

    2015-01-01

    Serine proteases perform proteolytic reactions in many physiological and metabolic processes and have been certified as targets for therapeutics. Small peptides can be used as potent antagonists to target serine proteases and intervene with their activities. Urokinase-type plasminogen activator (u......PA) plays an important role in plasminogen activation system, which has many physiological and pathological functions and is closely associated with the metastasis of tumor cells. Based on a mono-cyclic peptidic inhibitor of murine uPA (muPA), mupain-1, which was screened out from a phage-display library...... before, we elucidated the binding and inhibitory mechanism by using multiple techniques, like X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and surface plasmon resonance analysis. By studying the peptide-enzyme interaction, we discovered an unusual inhibitor...

  10. Evolution of microbial pathogens

    National Research Council Canada - National Science Library

    DiRita, Victor J; Seifert, H. Steven

    2006-01-01

    ... A. Hogan vvi ■ CONTENTS 8. Evolution of Pathogens in Soil Rachel Muir and Man-Wah Tan / 131 9. Experimental Models of Symbiotic Host-Microbial Relationships: Understanding the Underpinnings of ...

  11. Validation of two ribosomal RNA removal methods for microbial metatranscriptomics

    Energy Technology Data Exchange (ETDEWEB)

    He, Shaomei; Wurtzel, Omri; Singh, Kanwar; Froula, Jeff L; Yilmaz, Suzan; Tringe, Susannah G; Wang, Zhong; Chen, Feng; Lindquist, Erika A; Sorek, Rotem; Hugenholtz, Philip

    2010-10-01

    The predominance of rRNAs in the transcriptome is a major technical challenge in sequence-based analysis of cDNAs from microbial isolates and communities. Several approaches have been applied to deplete rRNAs from (meta)transcriptomes, but no systematic investigation of potential biases introduced by any of these approaches has been reported. Here we validated the effectiveness and fidelity of the two most commonly used approaches, subtractive hybridization and exonuclease digestion, as well as combinations of these treatments, on two synthetic five-microorganism metatranscriptomes using massively parallel sequencing. We found that the effectiveness of rRNA removal was a function of community composition and RNA integrity for these treatments. Subtractive hybridization alone introduced the least bias in relative transcript abundance, whereas exonuclease and in particular combined treatments greatly compromised mRNA abundance fidelity. Illumina sequencing itself also can compromise quantitative data analysis by introducing a G+C bias between runs.

  12. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis

    OpenAIRE

    Elsholz, Alexander K. W.; Birk, Marlene S.; Charpentier, Emmanuelle; Turgay, K?r?ad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor pro...

  13. Schistosome serine protease inhibitors: parasite defense or homeostasis?

    Directory of Open Access Journals (Sweden)

    Landys A. Lopez Quezada

    2011-06-01

    Full Text Available Serpins are a structurally conserved family of macromolecular inhibitors found in numerous biological systems. The completion and annotation of the genomes of Schistosoma mansoni and Schistosoma japonicum has enabled the identification by phylogenetic analysis of two major serpin clades. S. mansoni shows a greater multiplicity of serpin genes, perhaps reflecting adaptation to infection of a human host. Putative targets of schistosome serpins can be predicted from the sequence of the reactive center loop (RCL. Schistosome serpins may play important roles in both post-translational regulation of schistosome-derived proteases, as well as parasite defense mechanisms against the action of host proteases.Serpinas são uma família de inibidores macromoleculares estruturalmente conservados encontrados em inúmeros sistemas biológicos. O término e a anotação dos genomas de Schistosoma mansoni e de Schistosoma japonicum permitiram a identificação por análise filogenética de dois principais clados de serpinas. S. mansoni mostra uma multiplicidade maior de genes de serpinas, talvez refletindo uma adaptação à infecção de um hospedeiro humano. Alvos putativos das serpinas de esquistossomos podem ser preditos a partir da sequência do "loop" do centro reativo. Serpinas de esquistossomos podem ter importantes papeis tanto na regulação pós-traducional de proteases derivadas do esquistossoma, quanto nos mecanismos de defesa contra a ação de proteases do hospedeiro.

  14. Characterization and Preparation of Broken Rice Proteins Modified by Proteases

    Directory of Open Access Journals (Sweden)

    Lixia Hou

    2010-01-01

    Full Text Available Broken rice is an underutilized by-product of milling. Proteins prepared from broken rice by treatments with alkaline protease and papain have been characterized with regard to nutritional and functional properties. The protein content and the protein recovery were 56.45 and 75.45 % for alkaline protease treatment, and 65.45 and 46.32 % for papain treatment, respectively. Protease treatment increased the lysine and valine content, leading to a more balanced amino acid profile. Broken rice proteins had high emulsifying capacity, 58.3–71.6 % at neutral pH, and adequate water holding capacity, ranging from 1.96 to 2.93 g/g of proteins. At pH=7.0, the broken rice protein had the highest water holding capacity and the best interfacial activities (emulsifying capacity, emulsifying stability, foaming capacity and foaming stability, which may be the result of the higher solubility at pH=7.0. The interfacial activities increased with the increase in the mass fraction of broken rice proteins. The proteins prepared by the papain treatment had higher water holding capacity (p>0.05, emulsifying capacity (p0.05 than alkaline protease treatment at the same pH or mass fraction. To test the fortification of food products with broken rice proteins, pork sausages containing the proteins were prepared. Higher yield of the sausages was obtained with the increased content of broken rice proteins, in the range of 2.0–9.0 %. The results indicate that broken rice proteins have potential to be used as the protein fortification ingredient for food products.

  15. Viagra warning re "poppers" and notice re protease inhibitors.

    Science.gov (United States)

    James, J S

    1998-05-01

    Pfizer, Inc., manufacturer of Viagra (sildenafil), is warning patients that Viagra is contraindicated with nitrate inhalers (poppers). Nitrates in any form or nitric oxide donors can potentially cause hypotension. Pfizer also recommends that patients using protease inhibitors, particularly ritonavir, use a lower dose of Viagra (25 mg) because they interfere with the elimination of Viagra. This interaction does not pose a significant risk to the patient.

  16. Allicin and derivates are cysteine protease inhibitors with antiparasitic activity.

    Science.gov (United States)

    Waag, Thilo; Gelhaus, Christoph; Rath, Jennifer; Stich, August; Leippe, Matthias; Schirmeister, Tanja

    2010-09-15

    Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure-activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  17. Metabolic complications associated with HIV protease inhibitor therapy.

    Science.gov (United States)

    Nolan, David

    2003-01-01

    HIV protease inhibitors were introduced into clinical practice over 7 years ago as an important component of combination antiretroviral drug regimens which in many ways revolutionised the treatment of HIV infection. The significant improvements in prognosis that have resulted from the use of these regimens, combined with the need for lifelong treatment, have increasingly focused attention on the adverse effects of antiretroviral drugs and on the metabolic complications of HIV protease inhibitors in particular. In this review, the cluster of metabolic abnormalities characterised by triglyceride-rich dyslipidaemia and insulin resistance associated with HIV protease inhibitor therapy are considered, along with implications for cardiovascular risk in patients affected by these complications. Toxicity profiles of individual drugs within the HIV protease inhibitor class are examined, as there is an increased recognition of significant intra-class differences both in terms of absolute risk of metabolic complications as well as the particular metabolic phenotype associated with these drugs. Guidelines for clinical assessment and treatment are emphasised, along with pathophysiological mechanisms that may provide a rational basis for the treatment of metabolic complications. Finally, these drug-specific effects are considered within the context of HIV-specific effects on lipid metabolism as well as lifestyle factors that have contributed to a rapidly increasing incidence of similar metabolic syndromes in the general population. These data highlight the importance of individualising patient management in terms of choice of antiretroviral regimen, assessment of metabolic outcomes and use of therapeutic interventions, based on the assessment of baseline (pre-treatment) metabolic status as well as the presence of potentially modifiable cardiovascular risk factors.

  18. Rhomboid protease inhibitors: Emerging tools and future therapeutics

    Czech Academy of Sciences Publication Activity Database

    Stříšovský, Kvido

    2016-01-01

    Roč. 60, Dec (2016), s. 52-62 ISSN 1084-9521 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 EU Projects: European Commission(XE) 304154 - Rhomboid substrates Institutional support: RVO:61388963 Keywords : rhomboid protease * inhibitor * disease * mechanism * substrate specificity Subject RIV: CE - Biochemistry Impact factor: 6.614, year: 2016 http://www.sciencedirect.com/science/article/pii/S1084952116302592

  19. Evolutionary dynamics of hepatitis C virus NS3 protease domain during and following treatment with narlaprevir, a potent NS3 protease inhibitor

    NARCIS (Netherlands)

    de Bruijne, J.; Thomas, X. V.; Rebers, S. P.; Weegink, C. J.; Treitel, M. A.; Hughes, E.; Bergmann, J. F.; de Knegt, R. J.; Janssen, H. L. A.; Reesink, H. W.; Molenkamp, R.; Schinkel, J.

    2013-01-01

    Narlaprevir, a hepatitis C virus (HCV) NS3/4A serine protease inhibitor, has demonstrated robust antiviral activity in a placebo-controlled phase 1 study. To study evolutionary dynamics of resistant variants, the NS3 protease sequence was clonally analysed in thirty-two HCV genotype 1-infected

  20. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    Science.gov (United States)

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

    Zymographic analysis of Broccoli florets (Brassica oleracea L. var. Italica) revealed the presence of acidic metallo-proteases, serine proteases and cysteine proteases. Under conditions which were denaturing for the other proteases, the study was restricted to cysteine proteases. 2-D zymography, a technique that combines IEF and zymography was used to show the presence of 11 different cysteine protease spots with molecular mass of 44 and 47-48kDa and pIs ranging between 4.1 and 4.7. pI differences could be ascribed to different degrees of phosphorylation that partly disappeared in the presence of alkaline phosphatase. Post-harvest senescence of Broccoli florets was characterized by decrease in protein and chlorophyll contents and increase of protease activity. In particular, as determined by 2-D zymography, the presence of cysteine protease clearly increased during senescence, a finding that may represent a useful tool for the control of the aging process. Copyright © 2011 Elsevier GmbH. All rights reserved.

  1. Synthetic Electric Microbial Biosensors

    Science.gov (United States)

    2017-06-10

    domains and DNA-binding domains into a single protein for deregulation of down stream genes of have been favored [10]. Initially experiments with... Germany DISTRIBUTION A. Approved for public release: distribution unlimited.   Talk title: “Synthetic biology based microbial biosensors for the...toolbox” in Heidelberg, Germany Poster title: “Anaerobic whole cell microbial biosensors” Link: http://phdsymposium.embl.org/#home   September, 2014

  2. ISOLASI DAN KARAKTERISASI PROTEASE ALKALIN DARI ISOLAT BAKTERI LIMBAH TERNAK DI EXFARM FAKULTAS PETERNAKAN UNSOED

    Directory of Open Access Journals (Sweden)

    Zusfahair

    2011-05-01

    Full Text Available Protease is one of the widely used enzymes for the industry. The potential resource of microorganism that produced protease is milk cow waste. In this research, isolation and characterization has been done toward isolated protease from milk cow waste of the Exfarm’s Animal Husbandry Faculty at University of Jenderal Soedirman, Purwokerto. The research used experiment method and the parameters observed were the genus of bacteria which produce protease and the activity of protease. The characterizations of protease were determination of optimum pH and temperature, the influence of metal ions, EDTA, surfactant, and commercial detergent toward enzyme activity, and also the study of enzyme stability. The results from the research showed that the isolated bacteria from the Exfarm’s of Animal Husbandry Faculty of UNSOED, which produced protease was Salmonella sp. Characterization of isolated Salmonella sp. from 45% ammonium sulphate fraction indicated that the optimum temperature was 50 ºC, optimum pH was 8, the enzyme was activated by Ca2+ dan Mg2+ ion, whereas it was inhibited by Zn2+, Cu2+ ions and EDTA. The addition of Tween-80 with the concentration of 0.2% and 0.4% increased protease activity, however the addition of Tween-80 with concentration higher than 0.6% decreased the protease activity. Enzyme protease from isolated Salmonella sp. was relatively stable with the addition of commercial detergent such as Attack, Surf, and Bukrim.

  3. Enterovirus type 71 2A protease functions as a transcriptional activator in yeast

    Directory of Open Access Journals (Sweden)

    Lai Meng-Jiun

    2010-08-01

    Full Text Available Abstract Enterovirus type 71 (EV71 2A protease exhibited strong transcriptional activity in yeast cells. The transcriptional activity of 2A protease was independent of its protease activity. EV71 2A protease retained its transcriptional activity after truncation of 40 amino acids at the N-terminus but lost this activity after truncation of 60 amino acids at the N-terminus or deletion of 20 amino acids at the C-terminus. Thus, the acidic domain at the C-terminus of this protein is essential for its transcriptional activity. Indeed, deletion of amino acids from 146 to 149 (EAME in this acidic domain lost the transcriptional activity of EV71 2A protein though still retained its protease activity. EV71 2A protease was detected both in the cytoplasm and nucleus using confocal microscopy analysis. Coxsackie virus B3 2A protease also exhibited transcriptional activity in yeast cells. As expected, an acidic domain in the C-terminus of Coxsackie virus B3 2A protease was also identified. Truncation of this acidic domain resulted in the loss of transcriptional activity. Interestingly, this acidic region of poliovirus 2A protease is critical for viral RNA replication. The transcriptional activity of the EV71 or Coxsackie virus B3 2A protease should play a role in viral replication and/or pathogenesis.

  4. Cysteine proteases: Modes of activation and future prospects as pharmacological targets

    Directory of Open Access Journals (Sweden)

    Sonia eVerma

    2016-04-01

    Full Text Available Proteolytic enzymes are crucial for a variety of biological processes in organisms ranging from lower (virus, bacteria and parasite to the higher organisms (mammals. Proteases cleave proteins into smaller fragments by catalyzing peptide bonds hydrolysis. Proteases are classified according to their catalytic site, and distributed into four major classes: cysteine proteases, serine proteases, aspartic proteases and metallo-proteases. This review will cover only cysteine proteases, papain family enzymes which are involved in multiple functions such as extracellular matrix turnover, antigen presentation, processing events, digestion, immune invasion, hemoglobin hydrolysis, parasite invasion, parasite egress and processing surface proteins. Therefore, they are promising drug targets for various diseases. For preventing unwanted digestion, cysteine proteases are synthesized as zymogens, and contain a pro-domain (regulatory and a mature domain (catalytic. The prodomain acts as an endogenous inhibitor of the mature enzyme. For activation of the mature enzyme, removal of the prodomain is necessary and achieved by different modes. The pro-mature domain interaction can be categorized as protein-protein interactions (PPIs and may be targeted in a range of diseases. Cysteine protease inhibitors are available that can block the active site but no such inhibitor available yet that can be targeted to block the pro-mature domain interactions and prevent it activation. This review specifically highlights the modes of activation (processing of papain family enzymes, which involve auto-activation, trans-activation and also clarifies the future aspects of targeting PPIs to prevent the activation of cysteine proteases.

  5. Characterization of a membrane-associated serine protease in Escherichia coli

    International Nuclear Information System (INIS)

    Palmer, S.M.; St John, A.C.

    1987-01-01

    Three membrane-associated proteolytic activities in Escherichia coli were resolved by DEAE-cellulose chromatography from detergent extracts of the total envelope fraction. On the basis of substrate specificity for the hydrolysis of chromogenic amino acid ester substrates, the first two eluting activities were determined previously to be protease V and protease IV, respectively. The third proteolytic activity eluting from the DEAE-cellulose column was further purified by affinity chromatography on benzamidine-Sepharose 6B. They termed this enzyme protease VI. Protease VI did not hydrolyze any of the chromogenic substrates used in the detection of protease IV and protease V. However, all three enzymes generated acid-soluble fragments from a mixture of E. coli membrane proteins which were biosynthetically labeled with radioactive amino acids. The activity of protease VI was sensitive to serine protease inhibitors. Using [ 3 H]diisopropylfluorophosphate as an active-site labeling reagent, they determined that protease VI has an apparent molecular weight of 43,000 in polyacrylamide gels. All three membrane-associated serine proteases were insensitive to inhibition by Ecotin, an endogenous, periplasmic inhibitor of trypsin

  6. Autoprocessing of human immunodeficiency virus type 1 protease miniprecursor fusions in mammalian cells

    Directory of Open Access Journals (Sweden)

    Chen Chaoping

    2010-07-01

    Full Text Available Abstract Background HIV protease (PR is a virus-encoded aspartic protease that is essential for viral replication and infectivity. The fully active and mature dimeric protease is released from the Gag-Pol polyprotein as a result of precursor autoprocessing. Results We here describe a simple model system to directly examine HIV protease autoprocessing in transfected mammalian cells. A fusion precursor was engineered encoding GST fused to a well-characterized miniprecursor, consisting of the mature protease along with its upstream transframe region (TFR, and small peptide epitopes to facilitate detection of the precursor substrate and autoprocessing products. In HEK 293T cells, the resulting chimeric precursor undergoes effective autoprocessing, producing mature protease that is rapidly degraded likely via autoproteolysis. The known protease inhibitors Darunavir and Indinavir suppressed both precursor autoprocessing and autoproteolysis in a dose-dependent manner. Protease mutations that inhibit Gag processing as characterized using proviruses also reduced autoprocessing efficiency when they were introduced to the fusion precursor. Interestingly, autoprocessing of the fusion precursor requires neither the full proteolytic activity nor the majority of the N-terminal TFR region. Conclusions We suggest that the fusion precursors provide a useful system to study protease autoprocessing in mammalian cells, and may be further developed for screening of new drugs targeting HIV protease autoprocessing.

  7. Humanized-VHH Transbodies that Inhibit HCV Protease and Replication

    Directory of Open Access Journals (Sweden)

    Surasak Jittavisutthikul

    2015-04-01

    Full Text Available There is a need for safe and broadly effective anti-HCV agents that can cope with genetic multiplicity and mutations of the virus. In this study, humanized-camel VHHs to genotype 3a HCV serine protease were produced and were linked molecularly to a cell penetrating peptide, penetratin (PEN. Human hepatic (Huh7 cells transfected with the JFH-1 RNA of HCV genotype 2a and treated with the cell penetrable nanobodies (transbodies had a marked reduction of the HCV RNA intracellularly and in their culture fluids, less HCV foci inside the cells and less amounts of HCV core antigen in culture supernatants compared with the infected cells cultured in the medium alone. The PEN-VHH-treated-transfected cells also had up-regulation of the genes coding for the host innate immune response (TRIF, TRAF3, IRF3, IL-28B and IFN-β, indicating that the cell penetrable nanobodies rescued the host innate immune response from the HCV mediated-suppression. Computerized intermolecular docking revealed that the VHHs bound to residues of the protease catalytic triad, oxyanion loop and/or the NS3 N-terminal portion important for non-covalent binding of the NS4A protease cofactor protein. The so-produced transbodies have high potential for testing further as a candidate for safe, broadly effective and virus mutation tolerable anti-HCV agents.

  8. Detection of extracellular proteases from microorganisms on agar plates

    Directory of Open Access Journals (Sweden)

    Alane Beatriz Vermelho

    1996-12-01

    Full Text Available We present herein an improved assay for detecting the presence of extracellular proteases from microorganisms on agar plates. Using different substrates (gelatin, BSA, hemoglobin incorporated into the agar and varying the culture medium composition, we were able to detect proteolytic activities from Pseudomonas aeruginosa, Micrococcus luteus and Serratia marcescens as well as the influence that these components displayed in the expression of these enzymes. For all microorganisms tested we found that in agar-BHI or yeast extract medium containing gelatin the sensitivity of proteinase detection was considerably greater than in BSA-agar or hemoglobin-agar. However, when BSA or hemoglobin were added to the culture medium, there was an increase in growth along with a marked reduction in the amount of proteinase production. In the case of M. luteus the incorporation of glycerol in BHI or yeast extract gelatin-agar induced protease liberation. Our results indicate that the technique described here is of value for detecting extracellular proteases directly in the culture medium, by means of a qualitative assay, simple, inexpensive, straight forward method to assess the presence of the proteolytic activity of a given microorganism colony with great freedom in substrate selection.

  9. Protease activated receptors (PARS) mediation in gyroxin biological activity

    International Nuclear Information System (INIS)

    Silva, Jose Alberto Alves da

    2009-01-01

    Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH 2 , respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

  10. Targeting cysteine proteases in trypanosomatid disease drug discovery.

    Science.gov (United States)

    Ferreira, Leonardo G; Andricopulo, Adriano D

    2017-12-01

    Chagas disease and human African trypanosomiasis are endemic conditions in Latin America and Africa, respectively, for which no effective and safe therapy is available. Efforts in drug discovery have focused on several enzymes from these protozoans, among which cysteine proteases have been validated as molecular targets for pharmacological intervention. These enzymes are expressed during the entire life cycle of trypanosomatid parasites and are essential to many biological processes, including infectivity to the human host. As a result of advances in the knowledge of the structural aspects of cysteine proteases and their role in disease physiopathology, inhibition of these enzymes by small molecules has been demonstrated to be a worthwhile approach to trypanosomatid drug research. This review provides an update on drug discovery strategies targeting the cysteine peptidases cruzain from Trypanosoma cruzi and rhodesain and cathepsin B from Trypanosoma brucei. Given that current chemotherapy for Chagas disease and human African trypanosomiasis has several drawbacks, cysteine proteases will continue to be actively pursued as valuable molecular targets in trypanosomatid disease drug discovery efforts. Copyright © 2017. Published by Elsevier Inc.

  11. Pathogen-secreted proteases activate a novel plant immune pathway.

    Science.gov (United States)

    Cheng, Zhenyu; Li, Jian-Feng; Niu, Yajie; Zhang, Xue-Cheng; Woody, Owen Z; Xiong, Yan; Djonović, Slavica; Millet, Yves; Bush, Jenifer; McConkey, Brendan J; Sheen, Jen; Ausubel, Frederick M

    2015-05-14

    Mitogen-activated protein kinase (MAPK) cascades play central roles in innate immune signalling networks in plants and animals. In plants, however, the molecular mechanisms of how signal perception is transduced to MAPK activation remain elusive. Here we report that pathogen-secreted proteases activate a previously unknown signalling pathway in Arabidopsis thaliana involving the Gα, Gβ, and Gγ subunits of heterotrimeric G-protein complexes, which function upstream of an MAPK cascade. In this pathway, receptor for activated C kinase 1 (RACK1) functions as a novel scaffold that binds to the Gβ subunit as well as to all three tiers of the MAPK cascade, thereby linking upstream G-protein signalling to downstream activation of an MAPK cascade. The protease-G-protein-RACK1-MAPK cascade modules identified in these studies are distinct from previously described plant immune signalling pathways such as that elicited by bacterial flagellin, in which G proteins function downstream of or in parallel to an MAPK cascade without the involvement of the RACK1 scaffolding protein. The discovery of the new protease-mediated immune signalling pathway described here was facilitated by the use of the broad host range, opportunistic bacterial pathogen Pseudomonas aeruginosa. The ability of P. aeruginosa to infect both plants and animals makes it an excellent model to identify novel immunoregulatory strategies that account for its niche adaptation to diverse host tissues and immune systems.

  12. Microbial bioinformatics 2020.

    Science.gov (United States)

    Pallen, Mark J

    2016-09-01

    Microbial bioinformatics in 2020 will remain a vibrant, creative discipline, adding value to the ever-growing flood of new sequence data, while embracing novel technologies and fresh approaches. Databases and search strategies will struggle to cope and manual curation will not be sustainable during the scale-up to the million-microbial-genome era. Microbial taxonomy will have to adapt to a situation in which most microorganisms are discovered and characterised through the analysis of sequences. Genome sequencing will become a routine approach in clinical and research laboratories, with fresh demands for interpretable user-friendly outputs. The "internet of things" will penetrate healthcare systems, so that even a piece of hospital plumbing might have its own IP address that can be integrated with pathogen genome sequences. Microbiome mania will continue, but the tide will turn from molecular barcoding towards metagenomics. Crowd-sourced analyses will collide with cloud computing, but eternal vigilance will be the price of preventing the misinterpretation and overselling of microbial sequence data. Output from hand-held sequencers will be analysed on mobile devices. Open-source training materials will address the need for the development of a skilled labour force. As we boldly go into the third decade of the twenty-first century, microbial sequence space will remain the final frontier! © 2016 The Author. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  13. Studies on detection and analysis of proteases in leaf extract of medicinally important plants.

    Science.gov (United States)

    Chinnadurai, Gandhi Shree; Krishnan, Sivakumar; Perumal, Palani

    2018-02-01

    The whole plant or the extracts obtained from them have long been used as medicine to treat various human diseases and disorders. Notably, those plants endowed with protease activity have been traditionally used as the agents for treating tumors, digestion disorders, swelling, blood coagulation, fibrinolysis and also for immune-modulation. Proteases occupy a pivotal position in enzyme based industries. Plant proteases have been increasingly exploited for pharmaceutical, food, leather and textile processing industries. Earlier investigations have focused on the occurrence of proteases in medicinally unimportant plants. Therefore it has been aimed to study the occurrence of proteolytic enzymes from medicinally important plants establish any correlation exists between protease activity and medicinal use of individual plants. Crude extract were obtained from the leaves of 80 different medicinal plants. Tris-HCl buffer was used as the extraction buffer and the supernatants obtained were used for determination of total protein and protease activity using spectrophotometric methods. Qualitative screening for the presence of protease was carried out with agar diffusion method by incorporating the substrate. SDS-PAGE was used to analyse the isoforms of protease and for determination of relative molecular mass. Relatively higher protease activities were observed in the extracts of leaves of Pongamia pinnata (Fabaceae), Wrightia tinctoria (Apocyanaceae) Acalypha indica (Euphorbiaceae), Adhatoda vasica (Acanthaceae) and Curcuma longa (Zingiberaceae). No correlation was found between the total protein content and protease activity in individual plant species. SDS-PAGE analysis indicated the presence of multiple forms of protease of higher molecular weight range in several plant species. We found a strong correlation between the protease activity and medicinal application of the plant CONCLUSION: The present study has unequivocally revealed that the leaves of medicinal plants

  14. Resale Price Maintenance Under the Hong Kong Competition Ordinance—An Uneasy Compromise

    OpenAIRE

    Mark Jephcott; Adelaide Luke; Lisa Geary; Molly Herron

    2015-01-01

    However, the compromise position reached in the Guideline provides relatively little in terms of legal certainty as to the circumstances in which RPM may be acceptable. Mark Jephcott, Adelaide Luke, Lisa Geary, & Molly Herron (Herbert Smith Freehills)

  15. 48 CFR 239.7102-2 - Compromising emanations-TEMPEST or other standard.

    Science.gov (United States)

    2010-10-01

    ... INFORMATION TECHNOLOGY Security and Privacy for Computer Systems 239.7102-2 Compromising emanations—TEMPEST or... requiring activity is responsible for providing to the contracting officer— (a) The required protections, i...

  16. Small intestine epithelial barrier function is compromised in pigs with low feed intake at weaning.

    NARCIS (Netherlands)

    Spreeuwenberg, M.A.; Verdonk, J.M.; Gaskins, H.R.; Verstegen, M.W.A.

    2001-01-01

    Compromising alterations in gastrointestinal architecture are common during the weaning transition of pigs. The relation between villous atrophy and epithelial barrier function at weaning is not well understood. This study evaluated in vitro transepithelial transport by Ussing metabolic chambers,

  17. The Effect of Exogenous Protease in Broiler Diets on the Apparent Ileal Digestibility of Amino Acids and on Protease Activity in Jejunum

    Directory of Open Access Journals (Sweden)

    Vojtěch Rada

    2016-01-01

    Full Text Available The objective of this study was to evaluate the effect of a mono-component commercial serine protease supplement in broiler diets on apparent ileal amino acid digestibility and protease activity. A total of 150 male (28 d old ROSS 308 were randomly placed into 30 battery pens and divided into 5 treatment groups with 6 replicates each. The experiment was performed for 7 days. Five dietary treatments were used: 2 standard protein diets without (SP and with protease (SP + P formulated 20.7 % CP, 2 lower-protein diets (19.9 % CP without (LP and with protease (LP + P and one lower‑protein diet with protease and with doubled rapeseed meal (RSM content (SP-RSM + P compared with the other treatments. Lower-protein diets were formulated with a 4 % decrease in the relative CP value compared with the standard protein diet. Enzyme protease was added to the diets at a concentration of 200 ppm (15,000 PROT units per kg. The diets contained 0.3 % Cr2O3 to facilitate the estimation of apparent AA digestibility and overall apparent ileal crude protein digestibility. Mono-component protease had no effect on apparent ileal AA digestibility or jejunum protease activity if diets contained the same level of RSM. The supplement of exogenous protease did not affect (P > 0.05 the apparent ileal AA digestibility coefficients if a higher RSM level was used. The CP level influenced (P < 0.05 only the coefficients of the apparent ileal AA digestibility of Pro and Arg. The RSM level (P < 0.01 had significant effects on protease activity in the jejunum.

  18. Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients.

    Science.gov (United States)

    Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard; Schemann, Michael

    2018-01-01

    The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to

  19. Knowledge is power: averting safety-compromising events in the OR.

    Science.gov (United States)

    Catalano, Kathleen

    2008-12-01

    Surgical procedures can be unpredictable, and safety-compromising events can jeopardize patient safety. Perioperative nurses should be watchful for factors that can contribute to safety-compromising events, as well as the errors that can follow, and know how to avert them if possible. Knowledge is power and increased awareness of patient safety issues and the resources that are available to both health care practitioners and consumers can help perioperative nurses ward off patient safety problems before they occur.

  20. Cyber indicators of compromise: a domain ontology for security information and event management

    Science.gov (United States)

    2017-03-01

    heuristics, mapping, and detection. CybOX is aimed at supporting a broad range of important cyber security domains to include [31]: • Digital...REPORT TYPE AND DATES COVERED Master’s thesis 4. TITLE AND SUBTITLE CYBER INDICATORS OF COMPROMISE: A DOMAIN ONTOLOGY FOR SECURITY INFORMATION AND...Distribution is unlimited. CYBER INDICATORS OF COMPROMISE: A DOMAIN ONTOLOGY FOR SECURITY INFORMATION AND EVENT MANAGEMENT Marsha D. Rowell

  1. Combating QR-Code-Based Compromised Accounts in Mobile Social Networks.

    Science.gov (United States)

    Guo, Dong; Cao, Jian; Wang, Xiaoqi; Fu, Qiang; Li, Qiang

    2016-09-20

    Cyber Physical Social Sensing makes mobile social networks (MSNs) popular with users. However, such attacks are rampant as malicious URLs are spread covertly through quick response (QR) codes to control compromised accounts in MSNs to propagate malicious messages. Currently, there are generally two types of methods to identify compromised accounts in MSNs: one type is to analyze the potential threats on wireless access points and the potential threats on handheld devices' operation systems so as to stop compromised accounts from spreading malicious messages; the other type is to apply the method of detecting compromised accounts in online social networks to MSNs. The above types of methods above focus neither on the problems of MSNs themselves nor on the interaction of sensors' messages, which leads to the restrictiveness of platforms and the simplification of methods. In order to stop the spreading of compromised accounts in MSNs effectively, the attacks have to be traced to their sources first. Through sensors, users exchange information in MSNs and acquire information by scanning QR codes. Therefore, analyzing the traces of sensor-related information helps to identify the compromised accounts in MSNs. This paper analyzes the diversity of information sending modes of compromised accounts and normal accounts, analyzes the regularity of GPS (Global Positioning System)-based location information, and introduces the concepts of entropy and conditional entropy so as to construct an entropy-based model based on machine learning strategies. To achieve the goal, about 500,000 accounts of Sina Weibo and about 100 million corresponding messages are collected. Through the validation, the accuracy rate of the model is proved to be as high as 87.6%, and the false positive rate is only 3.7%. Meanwhile, the comparative experiments of the feature sets prove that sensor-based location information can be applied to detect the compromised accounts in MSNs.

  2. Combating QR-Code-Based Compromised Accounts in Mobile Social Networks

    Directory of Open Access Journals (Sweden)

    Dong Guo

    2016-09-01

    Full Text Available Cyber Physical Social Sensing makes mobile social networks (MSNs popular with users. However, such attacks are rampant as malicious URLs are spread covertly through quick response (QR codes to control compromised accounts in MSNs to propagate malicious messages. Currently, there are generally two types of methods to identify compromised accounts in MSNs: one type is to analyze the potential threats on wireless access points and the potential threats on handheld devices’ operation systems so as to stop compromised accounts from spreading malicious messages; the other type is to apply the method of detecting compromised accounts in online social networks to MSNs. The above types of methods above focus neither on the problems of MSNs themselves nor on the interaction of sensors’ messages, which leads to the restrictiveness of platforms and the simplification of methods. In order to stop the spreading of compromised accounts in MSNs effectively, the attacks have to be traced to their sources first. Through sensors, users exchange information in MSNs and acquire information by scanning QR codes. Therefore, analyzing the traces of sensor-related information helps to identify the compromised accounts in MSNs. This paper analyzes the diversity of information sending modes of compromised accounts and normal accounts, analyzes the regularity of GPS (Global Positioning System-based location information, and introduces the concepts of entropy and conditional entropy so as to construct an entropy-based model based on machine learning strategies. To achieve the goal, about 500,000 accounts of Sina Weibo and about 100 million corresponding messages are collected. Through the validation, the accuracy rate of the model is proved to be as high as 87.6%, and the false positive rate is only 3.7%. Meanwhile, the comparative experiments of the feature sets prove that sensor-based location information can be applied to detect the compromised accounts in MSNs.

  3. GS-8374, a Prototype Phosphonate-Containing Inhibitor of HIV-1 Protease, Effectively Inhibits Protease Mutants with Amino Acid Insertions

    Czech Academy of Sciences Publication Activity Database

    Grantz Šašková, Klára; Kožíšek, Milan; Stray, K.; Jong de, D.; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; Nijhuis, M.; Cihlář, T.; Konvalinka, Jan

    2014-01-01

    Roč. 88, č. 6 (2014), s. 3586-3590 ISSN 0022-538X R&D Projects: GA ČR GAP207/11/1798 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : virus type-1 protease * antiviral activity * drug resistance Subject RIV: EE - Microbiology, Virology Impact factor: 4.439, year: 2014

  4. Thermodynamic and structural analysis of HIV protease resistance to darunavir - analysis of heavily mutated patient- derived HIV-1 proteases

    Czech Academy of Sciences Publication Activity Database

    Kožíšek, Milan; Lepšík, Martin; Grantz Šašková, Klára; Brynda, Jiří; Konvalinka, Jan; Řezáčová, Pavlína

    2014-01-01

    Roč. 281, č. 7 (2014), s. 1834-1847 ISSN 1742-464X R&D Projects: GA ČR GAP207/11/1798 Grant - others:OPPC(XE) CZ.2.16/3.1.00/24016 Institutional support: RVO:61388963 ; RVO:68378050 Keywords : enthropic contribution * HIV protease inhibitors * isothermal titration calorimetry * resistance mutation * X-ray crystallography Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2014

  5. The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

    Czech Academy of Sciences Publication Activity Database

    Dostál, Jiří; Brynda, Jiří; Hrušková-Heidingsfeldová, Olga; Pachl, Petr; Pichová, Iva; Řezáčová, Pavlína

    2012-01-01

    Roč. 27, č. 1 (2012), s. 160-165 ISSN 1475-6366 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945; GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : secreted aspartic protease * virulence factor * X-ray structure * candidiasis Subject RIV: CE - Biochemistry Impact factor: 1.495, year: 2012

  6. Proteases from Latex of Euphorbia spp. and Its Application on Milk Clot Formation

    Directory of Open Access Journals (Sweden)

    Fidia Fibriana

    2015-09-01

    Full Text Available Crude proteases were extracted from Euphorbiaceae family, i.e. E. milii var imperata, E. trigona, and E. maculata. Among those three crude proteases, the activity of protease from E. trigona was the highest (812.50 U/ml, whereas E. milii and E. maculata crude proteases activity were 298.60 U/ml and 95.80 U/ml, respectively. E. maculata protein concentration was the highest among those three crude enzymes (1.206 mg/ml. The optimum pH and temperature of the enzymes were pH 7.0, pH 6.0, pH 6.5 and 60 °C, 50 °C, and 50 °C, respectively. Crude protease from E. milii var imperata, E. trigona, and E. maculata retained proteolytic activity over a wide range of pH (5.0–9.0 and temperature (up to 65 °C with casein as substrate. All crude proteases showed milk clotting activity ranged from 0.58 U/ml to 1.01 U/ml. Thus, these crude proteases are potential to be applied in dairy industries. However, further study on enzyme purification and characterization are necessary to obtain high purity of proteases before its application.Protease kasar berhasil diekstrak dari tanaman family Euphorbiaceae, yaitu E. milii var imperata, E. trigona, dan E. maculata. Diantara ketiga protease tersebut, aktivitas protease tertinggi diperoleh dari E. trigona (812,50 U/ml, sedangkan aktivitas protease dari E. milii dan E. maculata adalah 298,60 U/ml dan 95,80 U/ml, berturut-turut. Konsentrasi total protein tertinggi terdapat pada protease kasar E. maculata (1,206 mg/ml. pH dan suhu optimum ketiga enzim tersebut adalah pH 7.0, pH 6.0, pH 6.5 dan suhu 60 °C, 50 °C, and 50 °C, berturut-turut. Protease kasar dari E. milii var imperata, E. trigona, dan E. maculata menunjukkan aktivitas proteolitik pada rentang pH 5.0–9.0 dan rentang suhu sampai 65 °C menggunakan kasein sebagai substrat. Semua protease kasar menunjukkan aktivitas penggumpalan susu dengan rentang dari 0,58 U/ml sampai 1,01 U/ml. Berdasarkan hasil yang diperoleh, protease kasar dari ketiga jenis tanaman ini

  7. Identification of novel malarial cysteine protease inhibitors using structure-based virtual screening of a focused cysteine protease inhibitor library.

    Science.gov (United States)

    Shah, Falgun; Mukherjee, Prasenjit; Gut, Jiri; Legac, Jennifer; Rosenthal, Philip J; Tekwani, Babu L; Avery, Mitchell A

    2011-04-25

    Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

  8. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    Directory of Open Access Journals (Sweden)

    Werner Smidt

    Full Text Available The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1 infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS. Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  9. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    Science.gov (United States)

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  10. Variability and resistance mutations in the hepatitis C virus NS3 protease in patients not treated with protease inhibitors

    Directory of Open Access Journals (Sweden)

    Luciana Bonome Zeminian

    2013-02-01

    Full Text Available The goal of treatment of chronic hepatitis C is to achieve a sustained virological response, which is defined as exhibiting undetectable hepatitis C virus (HCV RNA levels in serum following therapy for at least six months. However, the current treatment is only effective in 50% of patients infected with HCV genotype 1, the most prevalent genotype in Brazil. Inhibitors of the serine protease non-structural protein 3 (NS3 have therefore been developed to improve the responses of HCV-infected patients. However, the emergence of drug-resistant variants has been the major obstacle to therapeutic success. The goal of this study was to evaluate the presence of resistance mutations and genetic polymorphisms in the NS3 genomic region of HCV from 37 patients infected with HCV genotype 1 had not been treated with protease inhibitors. Plasma viral RNA was used to amplify and sequence the HCV NS3 gene. The results indicate that the catalytic triad is conserved. A large number of substitutions were observed in codons 153, 40 and 91; the resistant variants T54A, T54S, V55A, R155K and A156T were also detected. This study shows that resistance mutations and genetic polymorphisms are present in the NS3 region of HCV in patients who have not been treated with protease inhibitors, data that are important in determining the efficiency of this new class of drugs in Brazil.

  11. Deep subsurface microbial processes

    Science.gov (United States)

    Lovley, D.R.; Chapelle, F.H.

    1995-01-01

    Information on the microbiology of the deep subsurface is necessary in order to understand the factors controlling the rate and extent of the microbially catalyzed redox reactions that influence the geophysical properties of these environments. Furthermore, there is an increasing threat that deep aquifers, an important drinking water resource, may be contaminated by man's activities, and there is a need to predict the extent to which microbial activity may remediate such contamination. Metabolically active microorganisms can be recovered from a diversity of deep subsurface environments. The available evidence suggests that these microorganisms are responsible for catalyzing the oxidation of organic matter coupled to a variety of electron acceptors just as microorganisms do in surface sediments, but at much slower rates. The technical difficulties in aseptically sampling deep subsurface sediments and the fact that microbial processes in laboratory incubations of deep subsurface material often do not mimic in situ processes frequently necessitate that microbial activity in the deep subsurface be inferred through nonmicrobiological analyses of ground water. These approaches include measurements of dissolved H2, which can predict the predominant microbially catalyzed redox reactions in aquifers, as well as geochemical and groundwater flow modeling, which can be used to estimate the rates of microbial processes. Microorganisms recovered from the deep subsurface have the potential to affect the fate of toxic organics and inorganic contaminants in groundwater. Microbial activity also greatly influences 1 the chemistry of many pristine groundwaters and contributes to such phenomena as porosity development in carbonate aquifers, accumulation of undesirably high concentrations of dissolved iron, and production of methane and hydrogen sulfide. Although the last decade has seen a dramatic increase in interest in deep subsurface microbiology, in comparison with the study of

  12. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins.

    Science.gov (United States)

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D

    2016-07-01

    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  13. Effect of amino acids on the repression of alkaline protease synthesis in haloalkaliphilic Nocardiopsis dassonvillei

    Directory of Open Access Journals (Sweden)

    Amit K. Sharma

    2016-12-01

    Full Text Available A newly isolated salt-tolerant alkaliphilic actinomycete, Nocardiopsis dassonvillei strain OK-18 grows on mineral salts medium with glucose as carbon source. It also grows and produces protease with amino acids as sole carbon source. The synthesis of extracellular alkaline protease parallel to growth was repressible by substrate concentrations. The absolute production of the protease was delinked with growth under nutritional stress, as protease production was high, despite poor growth. When amino acids served as the sole source of carbon and nitrogen, the enzyme production was significantly controlled by the number of amino acids. Maximal protease production was achieved with proline, asparagine, tyrosine, alanine, methionine and valine as sole source of carbon and nitrogen in minimal medium. With the increasing number of different amino acids in the presence and absence of glucose, the protease production was synergistically lower as compared to complex medium.

  14. Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age.

    Science.gov (United States)

    Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn; Dallas, David C

    2017-06-01

    Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL -1 · d -1 and 0.454 μg · mL -1 · d -1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not

  15. Three Pairs of Protease-Serpin Complexes Cooperatively Regulate the Insect Innate Immune Responses*

    OpenAIRE

    Jiang, Rui; Kim, Eun-Hye; Gong, Ji-Hee; Kwon, Hyun-Mi; Kim, Chan-Hee; Ryu, Kyoung-Hwa; Park, Ji-Won; Kurokawa, Kenji; Zhang, Jinghai; Gubb, David; Lee, Bok-Luel

    2009-01-01

    Serpins are known to be necessary for the regulation of several serine protease cascades. However, the mechanisms of how serpins regulate the innate immune responses of invertebrates are not well understood due to the uncertainty of the identity of the serine proteases targeted by the serpins. We recently reported the molecular activation mechanisms of three serine protease-mediated Toll and melanin synthesis cascades in a large beetle, Tenebrio molitor. Here, we purified three novel serpins ...

  16. Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

    Science.gov (United States)

    Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

    2017-03-01

    Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  17. Microbial conversion technologies

    Energy Technology Data Exchange (ETDEWEB)

    Lau, P. [National Research Council of Canada, Ottawa, ON (Canada). Bioconversion and Sustainable Development

    2006-07-01

    Microbes are a biomass and an valuable resource. This presentation discussed microbial conversion technologies along with background information on microbial cells, their characteristics and microbial diversity. Untapped opportunities for microbial conversion were identified. Metagenomic and genome mining approaches were also discussed, as they can provide access to uncultivated or unculturable microorganisms in communal populations and are an unlimited resource for biocatalysts, novel genes and metabolites. Genome mining was seen as an economical approach. The presentation also emphasized that the development of microbial biorefineries would require significant insights into the relevant microorganisms and that biocatalysts were the ultimate in sustainability. In addition, the presentation discussed the natural fibres initiative for biochemicals and biomaterials. Anticipated outputs were identified and work in progress of a new enzyme-retting cocktail to provide diversity and/or consistency in fibre characteristics for various applications were also presented. It was concluded that it is necessary to leverage understanding of biological processes to produce bioproducts in a clean and sustainable manner. tabs., figs.

  18. Purification and characterization of an alkaline protease from Micrococcus sp. isolated from the South China Sea

    Science.gov (United States)

    Hou, Enling; Xia, Tao; Zhang, Zhaohui; Mao, Xiangzhao

    2017-04-01

    Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0-11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn2+. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.

  19. Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

    DEFF Research Database (Denmark)

    Guarino, Carla; Hamon, Yveline; Croix, Cécile

    2017-01-01

    cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites....... These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases...

  20. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    International Nuclear Information System (INIS)

    Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi

    2010-01-01

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  1. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    Energy Technology Data Exchange (ETDEWEB)

    Jin Xin [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Li Jufang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Huang Pingying [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Dong Xuyan [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Guo Lulu [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Yang Liang; Cao Yuancheng [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Wei Fang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Zhao Yuandi, E-mail: zydi@mail.hust.edu.c [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China)

    2010-07-15

    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63+-2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  2. Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease

    Science.gov (United States)

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C.; Chang, Kyeong-Ok

    2012-01-01

    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

  3. Characterization of detergent compatible protease from halophilic Virgibacillus sp. CD6.

    Science.gov (United States)

    Lam, Ming Quan; Nik Mut, Nik Nurhidayu; Thevarajoo, Suganthi; Chen, Sye Jinn; Selvaratnam, Chitra; Hussin, Huszalina; Jamaluddin, Haryati; Chong, Chun Shiong

    2018-02-01

    A halophilic bacterium, Virgibacillus sp. strain CD6, was isolated from salted fish and its extracellular protease was characterized. Protease production was found to be highest when yeast extract was used as nitrogen source for growth. The protease exhibited stability at wide range of salt concentration (0-12.5%, w/v), temperatures (20-60 °C), and pH (4-10) with maximum activity at 10.0% (w/v) NaCl, 60 °C, pH 7 and 10, indicating its polyextremophilicity. The protease activity was enhanced in the presence of Mg 2+ , Mn 2+ , Cd 2+ , and Al 3+ (107-122% relative activity), and with retention of activity > 80% for all of other metal ions examined (K + , Ca 2+ , Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ , and Fe 3+ ). Both PMSF and EDTA inhibited protease activity, denoting serine protease and metalloprotease properties, respectively. High stability (> 70%) was demonstrated in the presence of organic solvents and detergent constituents, and the extracellular protease from strain CD6 was also found to be compatible in commercial detergents. Proteinaceous stain removal efficacy revealed that crude protease of strain CD6 could significantly enhance the performance of commercial detergent. The protease from Virgibacillus sp. strain CD6 could serve as a promising alternative for various applications, especially in detergent industry.

  4. Hepatic S1P deficiency lowers plasma cholesterol levels in apoB-containing lipoproteins when LDLR function is compromised.

    Science.gov (United States)

    Basu, Debapriya; Huq, Afroza; Iqbal, Jahangir; Hussain, M Mahmood; Jiang, Xian-Cheng; Jin, Weijun

    2015-01-01

    Site-1 protease (S1P) is the key enzyme required for activation of the sterol regulatory element binding proteins (SREBPs) that govern lipid synthesis. While S1P has been speculated to influence plasma apoB-containing lipoprotein (Blp) metabolism, there has been little investigative work. LDL receptor (LDLR) is the major receptor for clearing plasma LDL cholesterol (LDL-c). Proprotein convertase subtilisin kexin type 9 (PCSK9) modulates LDL-c through post-translational degradation of the LDLR. A hepatic-specific knockdown (KD) of S1P was achieved using floxed S1P mouse models (S1P(f/f) and LDLR(-/-)S1P(f/f)) and hepatic expression of Cre recombinase. Lipids were measured in total plasma and size fractionated plasma using colorimetric assays. Realtime polymerase chain reaction, western blotting and ELISA were used to determine hepatic expression of key genes/protein. Plasmid mediated overexpression and siRNA mediated knockdown of genes were performed in mouse primary hepatocytes to determine the mechanistic basis of PCSK9 gene regulation. A hepatic-specific KD of S1P resulted in a 45 % and 38 % reduction in plasma total cholesterol and triglyceride levels, respectively. Hepatic S1P KD had a minimal effect on plasma Blp cholesterol (Blp-c) in S1P(f/f) mice, despite significantly reducing VLDL secretion. Notably, hepatic S1P KD decreased the LDL receptor (LDLR) mRNA expression by 50 %. However, the reduction in LDLR protein levels was less than that of mRNA expression, especially under fed conditions. Further assessment of hepatic S1P deficiency revealed that it increased LDLR protein stability in vivo. Mechanistically, hepatic S1P KD was shown to decrease the liver and plasma levels of the protein proprotein convertase subtilisin/kexin type 9 (PCSK9), which degrades LDLR protein. This effect was more prominent in the fed condition and sufficient to account for the discordance in LDLR mRNA and protein levels. Furthermore, hepatic S1P was shown to regulate PCSK9

  5. The HtrA-like serine protease PepD interacts with and modulates the Mycobacterium tuberculosis 35-kDa antigen outer envelope protein.

    Directory of Open Access Journals (Sweden)

    Mark J White

    2011-03-01

    Full Text Available Mycobacterium tuberculosis remains a significant global health concern largely due to its ability to persist for extended periods within the granuloma of the host. While residing within the granuloma, the tubercle bacilli are likely to be exposed to stress that can result in formation of aberrant proteins with altered structures. Bacteria encode stress responsive determinants such as proteases and chaperones to deal with misfolded or unfolded proteins. pepD encodes an HtrA-like serine protease and is thought to process proteins altered following exposure of M. tuberculosis to extra-cytoplasmic stress. PepD functions both as a protease and chaperone in vitro, and is required for aspects of M. tuberculosis virulence in vivo. pepD is directly regulated by the stress-responsive two-component signal transduction system MprAB and indirectly by extracytoplasmic function (ECF sigma factor SigE. Loss of PepD also impacts expression of other stress-responsive determinants in M. tuberculosis. To further understand the role of PepD in stress adaptation by M. tuberculosis, a proteomics approach was taken to identify binding proteins and possible substrates of this protein. Using subcellular fractionation, the cellular localization of wild-type and PepD variants was determined. Purified fractions as well as whole cell lysates from Mycobacterium smegmatis or M. tuberculosis strains expressing a catalytically compromised PepD variant were immunoprecipitated for PepD and subjected to LC-MS/MS analyses. Using this strategy, the 35-kDa antigen encoding a homolog of the PspA phage shock protein was identified as a predominant binding partner and substrate of PepD. We postulate that proteolytic cleavage of the 35-kDa antigen by PepD helps maintain cell wall homeostasis in Mycobacterium and regulates specific stress response pathways during periods of extracytoplasmic stress.

  6. Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

    Science.gov (United States)

    Díaz, M E; Rocha, G F; Kise, F; Rosso, A M; Guevara, M G; Parisi, M G

    2018-05-08

    Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 μmol l -1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 μmol l -1 . Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would

  7. Functional protease profiling for laboratory based diagnosis of invasive aspergillosis.

    Science.gov (United States)

    Sabbagh, Bassel; Costina, Victor; Buchheidt, Dieter; Reinwald, Mark; Neumaier, Michael; Findeisen, Peter

    2015-07-01

    Invasive aspergillosis (IA) remains difficult to diagnose in immunocompromised patients, because diagnostic criteria according to EORTC/MSG guidelines are often not met and have low sensitivity. Hence there is an urgent need to improve diagnostic procedures by developing novel approaches. In the present study, we present a proof of concept experiment for the monitoring of Aspergillus associated protease activity in serum specimens for diagnostic purpose. Synthetic peptides that are selectively cleaved by proteases secreted from Aspergillus species were selected from our own experiments and published data. These so called reporter peptides (RP, n=5) were added to serum specimens from healthy controls (HC, n=101) and patients with proven (IA, n=9) and possible (PIA, n=144) invasive aspergillosis. Spiked samples were incubated ex vivo under strictly standardized conditions. Proteolytic fragments were analyzed using MALDI-TOF mass spectrometry. Spiked specimens of IA patients had highest concentrations of RP-fragments followed by PIA and HC. The median signal intensity was 116.546 (SD, 53.063) for IA and 5.009 (SD, 8.432) for HC. A cut-off >36.910 was chosen that performed with 100% specificity and sensitivity. Patients with PIA had either values above [53% (76/144)] or below [47% (67/144)] this chosen cut-off. The detection of respective reporter peptide fragments can easily be performed by MALDI TOF mass spectrometry. In this proof of concept study we were able to demonstrate that serum specimens of patients with IA have increased proteolytic activity towards selected reporter peptides. However, the diagnostic value of functional protease profiling has to be validated in further prospective studies. It is likely that a combination of existing and new methods will be required to achieve optimal performance for diagnosis of IA in the future.

  8. EVA Suit Microbial Leakage Investigation

    Data.gov (United States)

    National Aeronautics and Space Administration — The objective of this project is to collect microbial samples from various EVA suits to determine how much microbial contamination is typically released during...

  9. Microbial Invasion vs. Tick Immune Regulation.

    Science.gov (United States)

    Sonenshine, Daniel E; Macaluso, Kevin R

    2017-01-01

    Ticks transmit a greater variety of pathogenic agents that cause disease in humans and animals than any other haematophagous arthropod, including Lyme disease, Rocky Mountain spotted fever, human granulocytic anaplasmosis, babesiosis, tick-borne encephalitis, Crimean Congo haemorhagic fever, and many others (Gulia-Nuss et al., 2016). Although diverse explanations have been proposed to explain their remarkable vectorial capacity, among the most important are their blood feeding habit, their long term off-host survival, the diverse array of bioactive molecules that disrupt the host's natural hemostatic mechanisms, facilitate blood flow, pain inhibitors, and minimize inflammation to prevent immune rejection (Hajdušek et al., 2013). Moreover, the tick's unique intracellular digestive processes allow the midgut to provide a relatively permissive microenvironment for survival of invading microbes. Although tick-host-pathogen interactions have evolved over more than 300 million years (Barker and Murrell, 2008), few microbes have been able to overcome the tick's innate immune system, comprising both humoral and cellular processes that reject them. Similar to most eukaryotes, the signaling pathways that regulate the innate immune response, i.e., the Toll, IMD (Immunodeficiency) and JAK-STAT (Janus Kinase/ Signal Transducers and Activators of Transcription) also occur in ticks (Gulia-Nuss et al., 2016). Recognition of pathogen-associated molecular patterns (PAMPs) on the microbial surface triggers one or the other of these pathways. Consequently, ticks are able to mount an impressive array of humoral and cellular responses to microbial challenge, including anti-microbial peptides (AMPs), e.g., defensins, lysozymes, microplusins, etc., that directly kill, entrap or inhibit the invaders. Equally important are cellular processes, primarily phagocytosis, that capture, ingest, or encapsulate invading microbes, regulated by a primordial system of thioester-containing proteins

  10. Microbial Invasion vs. Tick Immune Regulation

    Directory of Open Access Journals (Sweden)

    Daniel E. Sonenshine

    2017-09-01

    Full Text Available Ticks transmit a greater variety of pathogenic agents that cause disease in humans and animals than any other haematophagous arthropod, including Lyme disease, Rocky Mountain spotted fever, human granulocytic anaplasmosis, babesiosis, tick-borne encephalitis, Crimean Congo haemorhagic fever, and many others (Gulia-Nuss et al., 2016. Although diverse explanations have been proposed to explain their remarkable vectorial capacity, among the most important are their blood feeding habit, their long term off-host survival, the diverse array of bioactive molecules that disrupt the host's natural hemostatic mechanisms, facilitate blood flow, pain inhibitors, and minimize inflammation to prevent immune rejection (Hajdušek et al., 2013. Moreover, the tick's unique intracellular digestive processes allow the midgut to provide a relatively permissive microenvironment for survival of invading microbes. Although tick-host-pathogen interactions have evolved over more than 300 million years (Barker and Murrell, 2008, few microbes have been able to overcome the tick's innate immune system, comprising both humoral and cellular processes that reject them. Similar to most eukaryotes, the signaling pathways that regulate the innate immune response, i.e., the Toll, IMD (Immunodeficiency and JAK-STAT (Janus Kinase/ Signal Transducers and Activators of Transcription also occur in ticks (Gulia-Nuss et al., 2016. Recognition of pathogen-associated molecular patterns (PAMPs on the microbial surface triggers one or the other of these pathways. Consequently, ticks are able to mount an impressive array of humoral and cellular responses to microbial challenge, including anti-microbial peptides (AMPs, e.g., defensins, lysozymes, microplusins, etc., that directly kill, entrap or inhibit the invaders. Equally important are cellular processes, primarily phagocytosis, that capture, ingest, or encapsulate invading microbes, regulated by a primordial system of thioester

  11. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis.

    Science.gov (United States)

    Elsholz, Alexander K W; Birk, Marlene S; Charpentier, Emmanuelle; Turgay, Kürşad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis . We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics.

  12. Functional Diversity of AAA+ Protease Complexes in Bacillus subtilis

    Science.gov (United States)

    Elsholz, Alexander K. W.; Birk, Marlene S.; Charpentier, Emmanuelle; Turgay, Kürşad

    2017-01-01

    Here, we review the diverse roles and functions of AAA+ protease complexes in protein homeostasis, control of stress response and cellular development pathways by regulatory and general proteolysis in the Gram-positive model organism Bacillus subtilis. We discuss in detail the intricate involvement of AAA+ protein complexes in controlling sporulation, the heat shock response and the role of adaptor proteins in these processes. The investigation of these protein complexes and their adaptor proteins has revealed their relevance for Gram-positive pathogens and their potential as targets for new antibiotics. PMID:28748186

  13. Boceprevir: a protease inhibitor for the treatment of hepatitis C.

    Science.gov (United States)

    Chang, Mei H; Gordon, Lori A; Fung, Horatio B

    2012-10-01

    Boceprevir is a protease inhibitor indicated for the treatment of chronic hepatitis C virus (HCV) genotype 1 infection in combination with peginterferon and ribavirin for treatment-naive patients and those who previously failed to improve with interferon and ribavirin treatment. This article provides an overview of the mechanism of action, pharmacologic and pharmacokinetic properties, clinical efficacy, and tolerability of boceprevir. Relevant information was identified through a search of PubMed (1990-July 2012), EMBASE (1990-July 2012), International Pharmaceutical Abstracts (1970-July 2012), and Google Scholar using the key words boceprevir, SCH 503034, non-structural protein 3 (NS3) serine protease inhibitor, and direct-acting antiviral agent (DAA). Additional information was obtained from the US Food and Drug Administration's Web site, review of the reference lists of identified articles, and posters and abstracts from scientific meetings. Clinical efficacy of boceprevir was assessed in 2 Phase III trials, Serine Protease Inhibitor Therapy-2 (SPRINT-2) for treatment-naive patients and Retreatment with HCV Serine Protease Inhibitor Boceprevir and PegIntron/Rebetol 2 (RESPOND-2) for treatment-experienced patients. In SPRINT-2, patients were randomized to receive peginterferon + ribavirin (PR) or peginterferon + ribavirin + boceprevir (PRB); duration of boceprevir therapy varied from 24, 32, to 44 weeks on the basis of HCV RNA results. The primary endpoint was achievement of sustained virologic response (SVR; lower limit of detection, 9.3 IU/mL). The addition of boceprevir was shown to be superior, with overall SVR rates ranging from 63% to 66% compared with 38% with PR (P < 0.001). Results of SVR in SPRINT-2 were also reorganized to monitor SVRs in black and non-black patients. Treatment-experienced patients were assessed in RESPOND-2; however, null responders were excluded. Patients were again randomized to PR or PRB; duration of boceprevir therapy varied from

  14. Antiretroviral activity of protease inhibitors against Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Lianet Monzote

    2013-02-01

    Full Text Available The introduction of highly active antiretroviral therapy (HAART has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE. These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

  15. HIV-protease inhibitors for the treatment of cancer

    DEFF Research Database (Denmark)

    Maksimovic-Ivanic, Danijela; Fagone, Paolo; McCubrey, James

    2017-01-01

    The possible use of HIV protease inhibitors (HIV-PI) as new therapeutic option for the treatment of cancer primarily originated from their success in treating HIV-related Kaposi's sarcoma (KS). While these findings were initially attributed to immune reconstitution and better control of oncogenic...... and nitric oxide (NO) derivatives of HIV-PIs. In this article, we discuss the current preclinical and clinical evidences for the potential use of HIV-PIs, and of novel derivatives, such as saquinavir-NO in the treatment of cancer....

  16. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop?

    Science.gov (United States)

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique

    2016-08-01

    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

  17. An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease

    Directory of Open Access Journals (Sweden)

    Vikram Surendra

    2010-07-01

    Full Text Available Abstract Background Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min. Results Seventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+ and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin. Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C. Conclusions An antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.

  18. Molecular ecology of microbial mats

    NARCIS (Netherlands)

    Bolhuis, H.; Cretoiu, M.S.; Stal, L.J.

    2014-01-01

    Phototrophic microbial mats are ideal model systems for ecological and evolutionary analysis of highly diverse microbial communities. Microbial mats are small-scale, nearly closed, and self-sustaining benthic ecosystems that comprise the major element cycles, trophic levels, and food webs. The steep

  19. Prokaryotic Selectivity, Anti-endotoxic Activity and Protease Stability of Diastereomeric and Enantiomeric Analogs of Human Antimicrobial Peptide LL-37

    International Nuclear Information System (INIS)

    Nan, Yong Hai; Lee, Bongju; Shin, Song Yub

    2012-01-01

    LL-37 is the only antimicrobial peptide (AMP) of the human cathelicidin family. In addition to potent antimicrobial activity, LL-37 is known to have the potential to inhibit lipolysaccharide (LPS)-induced endotoxic effects. To provide the stability to proteolytic digestion and increase prokaryotic selectivity and/or anti-endotoxic activity of two Lys/Trp-substituted 19-meric anti-microbial peptides (a4-W1 and a4-W2) designed from IG-19 (residues 13-31 of LL-37), we synthesized the diastereomeric peptides (a4-W1-D and a4-W2-D) with D-amino acid substitution at positions 3, 7, 10, 13 and 17 of a4-W1 and a4-W2, respectively and the enantiomeric peptides (a4-W1-E and a4-W2-E) composed D-amino acids. The diastereomeric peptides exhibited the best prokaryotic selectivity and effective protease stability, but no or less anti-endotoxic activity. In contrast, the enantiomeric peptides had not only prokaryotic selectivity and anti-endotoxic activity but also protease stability. Our results suggest that the hydrophobicity and α-helicity of the peptide is important for anti-endotoxic activity. In particular, the enantiomeric peptides showed potent anti-endotoxic and LPS-neutralizing activities comparable to that of LL-37. Taken together, both a4-W1-E and a4-W2-E holds promise as a template for the development of peptide antibiotics for the treatment of endotoxic shock and sepsis

  20. Prokaryotic Selectivity, Anti-endotoxic Activity and Protease Stability of Diastereomeric and Enantiomeric Analogs of Human Antimicrobial Peptide LL-37

    Energy Technology Data Exchange (ETDEWEB)

    Nan, Yong Hai; Lee, Bongju; Shin, Song Yub [Chosun Univ., Gwangju (Korea, Republic of)

    2012-09-15

    LL-37 is the only antimicrobial peptide (AMP) of the human cathelicidin family. In addition to potent antimicrobial activity, LL-37 is known to have the potential to inhibit lipolysaccharide (LPS)-induced endotoxic effects. To provide the stability to proteolytic digestion and increase prokaryotic selectivity and/or anti-endotoxic activity of two Lys/Trp-substituted 19-meric anti-microbial peptides (a4-W1 and a4-W2) designed from IG-19 (residues 13-31 of LL-37), we synthesized the diastereomeric peptides (a4-W1-D and a4-W2-D) with D-amino acid substitution at positions 3, 7, 10, 13 and 17 of a4-W1 and a4-W2, respectively and the enantiomeric peptides (a4-W1-E and a4-W2-E) composed D-amino acids. The diastereomeric peptides exhibited the best prokaryotic selectivity and effective protease stability, but no or less anti-endotoxic activity. In contrast, the enantiomeric peptides had not only prokaryotic selectivity and anti-endotoxic activity but also protease stability. Our results suggest that the hydrophobicity and α-helicity of the peptide is important for anti-endotoxic activity. In particular, the enantiomeric peptides showed potent anti-endotoxic and LPS-neutralizing activities comparable to that of LL-37. Taken together, both a4-W1-E and a4-W2-E holds promise as a template for the development of peptide antibiotics for the treatment of endotoxic shock and sepsis.

  1. Quantitative assessment of in situ microbial communities affecting nuclear waste disposal

    International Nuclear Information System (INIS)

    White, D.C.

    1996-01-01

    Microbes in the environments surrounding nuclear waste depositories pose several questions regarding the protection of the surrounding communities. microbes can facilitate microbially influenced corrosion (MIC), mobilize and facilitate the transport of nuclides as well as produce gaseous emissions which can compromise containment. We have developed an analysis of the extant microbiota that is independent of quantitative recovery and subsequent growth, based on signature biomarkers analysis (SBA)

  2. Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

    1975-12-01

    The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  3. Anaerobic microbial dehalogenation

    NARCIS (Netherlands)

    Smidt, H.; Vos, de W.M.

    2004-01-01

    The natural production and anthropogenic release of halogenated hydrocarbons into the environment has been the likely driving force for the evolution of an unexpectedly high microbial capacity to dehalogenate different classes of xenobiotic haloorganics. This contribution provides an update on the

  4. Diazotrophic microbial mats

    NARCIS (Netherlands)

    Severin, I.; Stal, L.J.; Seckbach, J.; Oren, A.

    2010-01-01

    Microbial mats have been the focus of scientific research for a few decades. These small-scale ecosystems are examples of versatile benthic communities of microorganisms, usually dominated by phototrophic bacteria (e.g., Krumbein et al., 1977; Jørgensen et al., 1983). They develop as vertically

  5. Microbial Energy Conversion

    Energy Technology Data Exchange (ETDEWEB)

    Buckley, Merry [American Society for Microbiology (ASM), Washington, DC (United States); Wall, Judy D. [Univ. of Missouri, Columbia, MO (United States)

    2006-10-01

    The American Academy of Microbiology convened a colloquium March 10-12, 2006, in San Francisco, California, to discuss the production of energy fuels by microbial conversions. The status of research into various microbial energy technologies, the advantages and disadvantages of each of these approaches, research needs in the field, and education and training issues were examined, with the goal of identifying routes for producing biofuels that would both decrease the need for fossil fuels and reduce greenhouse gas emissions. Currently, the choices for providing energy are limited. Policy makers and the research community must begin to pursue a broader array of potential energy technologies. A diverse energy portfolio that includes an assortment of microbial energy choices will allow communities and consumers to select the best energy solution for their own particular needs. Funding agencies and governments alike need to prepare for future energy needs by investing both in the microbial energy technologies that work today and in the untested technologies that will serve the world’s needs tomorrow. More mature bioprocesses, such as ethanol production from starchy materials and methane from waste digestors, will find applications in the short term. However, innovative techniques for liquid fuel or biohydrogen production are among the longer term possibilities that should also be vigorously explored, starting now. Microorganisms can help meet human energy needs in any of a number of ways. In their most obvious role in energy conversion, microorganisms can generate fuels, including ethanol, hydrogen, methane, lipids, and butanol, which can be burned to produce energy. Alternatively, bacteria can be put to use in microbial fuel cells, where they carry out the direct conversion of biomass into electricity. Microorganisms may also be used some day to make oil and natural gas technologies more efficient by sequestering carbon or by assisting in the recovery of oil and

  6. Microbial electrosynthesis of biochemicals

    NARCIS (Netherlands)

    Bajracharya, S.

    2016-01-01

    Microbial electrosynthesis (MES) is an electricity-driven production of chemicals from low-value waste using microorganisms as biocatalysts. MES from CO2 comprises conversion of CO2 to multi-carbon compounds employing microbes at the cathode which use electricity as an energy source. This thesis

  7. Secapin, a bee venom peptide, exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities.

    Science.gov (United States)

    Lee, Kwang Sik; Kim, Bo Yeon; Yoon, Hyung Joo; Choi, Yong Soo; Jin, Byung Rae

    2016-10-01

    Bee venom contains a variety of peptide constituents that have various biological, toxicological, and pharmacological actions. However, the biological actions of secapin, a venom peptide in bee venom, remain largely unknown. Here, we provide the evidence that Asiatic honeybee (Apis cerana) secapin (AcSecapin-1) exhibits anti-fibrinolytic, anti-elastolytic, and anti-microbial activities. The recombinant mature AcSecapin-1 peptide was expressed in baculovirus-infected insect cells. AcSecapin-1 functions as a serine protease inhibitor-like peptide that has inhibitory effects against plasmin, elastases, microbial serine proteases, trypsin, and chymotrypsin. Consistent with these functions, AcSecapin-1 inhibited the plasmin-mediated degradation of fibrin to fibrin degradation products, thus indicating the role of AcSecapin-1 as an anti-fibrinolytic agent. AcSecapin-1 also inhibited both human neutrophil and porcine pancreatic elastases. Furthermore, AcSecapin-1 bound to bacterial and fungal surfaces and exhibited anti-microbial activity against fungi and gram-positive and gram-negative bacteria. Taken together, our data demonstrated that the bee venom peptide secapin has multifunctional roles as an anti-fibrinolytic agent during fibrinolysis and an anti-microbial agent in the innate immune response. Copyright © 2016 Elsevier Ltd. All rights reserved.

  8. The impact of purchase quantity on the compromise effect: The balance heuristic

    Directory of Open Access Journals (Sweden)

    Yin-Hui Cheng

    2012-07-01

    Full Text Available Most prior research on the compromise effect has focused on single rather than multiple choices. This research investigates the potential effects of purchase quantity on the compromise effect. We propose that the share of the middle option in a trinary choice set decreases as the purchase quantity increases, because people tend to employ a balance heuristic to distribute their multiple choices among the available options to achieve a balanced state and to satisfy their variety-seeking tendency. Furthermore, we propose that the need for justification and an optimal stimulation level moderate the relationship between the number of purchase items and the compromise effect. These proposed hypotheses are supported by results from three experiments.

  9. Robust Public Key Cryptography — A New Cryptosystem Surviving Private Key Compromise

    Science.gov (United States)

    Shaik, Cheman

    A weakness of the present-day public key cryptosystems is that these cryptosystems do not survive private-key compromise attacks resulting from an internal breach of trust. In a competitive business environment, private key compromise is a common incident that voids the strength of public key cryptosystems such as RSA and ECC. Bribing corporate employees to disclose their secret keys and inadvertently disclosing secret information are among a plethora of practical attacks that occur at the implementation level. Once a breach of trust takes place and subsequently the private key is revealed, any public key cryptosystem fails to secure electronic data in Internet communications. The revealed key may be used by an attacker to decipher the intercepted data at an intermediary router. This weakness of public key cryptography calls for an additional security measure that enables encryptions to survive private key compromise attacks.

  10. Orthodontic space closure using simple mechanics in compromised first molar extraction spaces: Case series

    Directory of Open Access Journals (Sweden)

    Prashant M Dhole

    2018-01-01

    Full Text Available Orthodontic treatments involving missing or compromised first permanent molars are often challenging cases to treat considering the loss of potential anchor tooth. The case series presents orthodontic treatment in three patients with mutilated/absent first permanent molars. The treatment goals were to be accomplished by extraction of compromised teeth and retention of healthy dentition. The diagnosis and problem list needed extractions to accomplish the treatment goals. The possibility of extracting compromised first permanent molars instead of other healthy teeth was considered. A setup was done to determine the final occlusion in asymmetric extraction cases. Fixed appliances were used with simple mechanics without any additional anchorage devices. Case-based retention protocols were followed. All the three patients achieved the predetermined treatment objectives of improved esthetics and healthy and stable functional occlusion. The simple and efficient mechanics and finishing techniques for comprehensive orthodontic treatment with first molar extractions presented in the report should help clinicians when treating similar patients.

  11. Does low self-esteem predict health compromising behaviours among adolescents?

    Science.gov (United States)

    Mcgee, R; Williams, S

    2000-10-01

    It is often believed that low self-esteem is associated with such health-compromising behaviours in adolescence as substance use, early sexual activity, eating problems and suicidal ideation. Surprisingly, there is little longitudinal research addressing this issue. This longitudinal study examines the predictive association between both global and academic self-esteem from ages 9 to 13 years, and a variety of health compromising behaviours at age 15, in a large sample of young New Zealanders. Levels of global self-esteem significantly predicted adolescent report of problem eating, suicidal ideation, and multiple health compromising behaviours. Earlier levels of self-esteem were unrelated to later substance use and early sexual activity. The findings are discussed in terms of their implications for efforts to raise self-esteem among young people. Copyright 2000 The Association for Professionals in Services for Adolescents.

  12. Rapid monitoring of autolysis process of proteases by capillary electrophoresis.

    Science.gov (United States)

    Chen, Xiu-Lan; Shun, Cai-Yun; Zhang, Yu-Zhong; Gao, Pei-Ji

    2003-10-01

    A protease, MCP-01, produced by a deep-sea psychrotrophic strain of Pseudoaltermonas sp. SM9913 was purified and its autolysis reaction at 20 degrees C-50 degrees C was monitored by capillary electrophoresis. Capillary electrophoresis provides a rapid assay because the degree and state of autolysis of protease MCP-01 could be observed within 6 min. The autolysis rate increased as the temperature rose in the tested range. After 30 min incubation at 30 degrees C, 77% of MCP-01 autolyzed into peptides. However, its activity for the hydrolysis of casein was reduced by only 4%. The rate of loss of activity of MCP-01 was thus slower than that of autolysis of MCP-01 at 30 degrees C. Similar results were obtained when MCP-01 was incubated at 20 degrees C, 40 degrees C and 50 degrees C. Large peptides produced by autolysis of MCP-01 therefore still have catalytic activity. When these large peptides autolyzed further into smaller peptides, the enzyme conformation that retained its catalytic activity was destroyed and activity was lost.

  13. Mast cell protease 6 is required for allograft tolerance.

    Science.gov (United States)

    de Vries, V C; Elgueta, R; Lee, D M; Noelle, R J

    2010-09-01

    It has been shown that mast cells (MC) are absolutely required for transplant acceptance. However, only a few of the numerous mediators produced by MC have been proposed as potential mechanisms for the observed immunosuppression. The role of proteases in acquired immune tolerance as such has not yet been addressed. In this study, we have shown the requirement for MC protease 6 (MCP6), an MC-specific tryptase, to establish tolerance toward an allogeneic skin graft. The substrate for MCP6 is interleukin (IL)-6, cytokine generally considered to indicate transplant rejection. Herein we have shown an inverse correlation between MCP6 and IL-6. High expression of MCP6 is accompanied by low levels of IL-6 when the allograft is accepted, whereas low expression of MCP6 in combination with high levels of IL-6 are observed in rejecting grafts. Moreover, tolerance toward an allogeneic graft cannot be induced in MCP6(-/-) mice. Rejection observed in these mice was comparable to that of MC-deficient hosts; it is T-cell mediated. These findings suggest that MCP6 actively depletes the local environment of IL-6 to maintain tolerance. 2010. Published by Elsevier Inc.

  14. Short hydrogen bonds in the catalytic mechanism of serine proteases

    Directory of Open Access Journals (Sweden)

    VLADIMIR LESKOVAC

    2008-04-01

    Full Text Available The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78–1.28 Å revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 Å long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short “low-barrier” hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the “low-barrier hydrogen bond” hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 Å long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the “low-barrier hydrogen bond” hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

  15. Epigenetic silencing of serine protease HTRA1 drives polyploidy

    International Nuclear Information System (INIS)

    Schmidt, Nina; Irle, Inga; Ripkens, Kamilla; Lux, Vanda; Nelles, Jasmin; Johannes, Christian; Parry, Lee; Greenow, Kirsty; Amir, Sarah; Campioni, Mara; Baldi, Alfonso; Oka, Chio; Kawaichi, Masashi; Clarke, Alan R.; Ehrmann, Michael

    2016-01-01

    Increased numbers and improperly positioned centrosomes, aneuploidy or polyploidy, and chromosomal instability are frequently observed characteristics of cancer cells. While some aspects of these events and the checkpoint mechanisms are well studied, not all players have yet been identified. As the role of proteases other than the proteasome in tumorigenesis is an insufficiently addressed question, we investigated the epigenetic control of the widely conserved protease HTRA1 and the phenotypes of deregulation. Mouse embryonal fibroblasts and HCT116 and SW480 cells were used to study the mechanism of epigenetic silencing of HTRA1. In addition, using cell biological and genetic methods, the phenotypes of downregulation of HTRA1 expression were investigated. HTRA1 is epigenetically silenced in HCT116 colon carcinoma cells via the epigenetic adaptor protein MBD2. On the cellular level, HTRA1 depletion causes multiple phenotypes including acceleration of cell growth, centrosome amplification and polyploidy in SW480 colon adenocarcinoma cells as well as in primary mouse embryonic fibroblasts (MEFs). Downregulation of HTRA1 causes a number of phenotypes that are hallmarks of cancer cells suggesting that the methylation state of the HtrA1 promoter may be used as a biomarker for tumour cells or cells at risk of transformation. The online version of this article (doi:10.1186/s12885-016-2425-8) contains supplementary material, which is available to authorized users

  16. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    Science.gov (United States)

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  18. MOFzyme: Intrinsic protease-like activity of Cu-MOF

    Science.gov (United States)

    Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

    2014-10-01

    The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

  19. Multiple roles of the coagulation protease cascade during virus infection.

    Science.gov (United States)

    Antoniak, Silvio; Mackman, Nigel

    2014-04-24

    The coagulation cascade is activated during viral infections. This response may be part of the host defense system to limit spread of the pathogen. However, excessive activation of the coagulation cascade can be deleterious. In fact, inhibition of the tissue factor/factor VIIa complex reduced mortality in a monkey model of Ebola hemorrhagic fever. Other studies showed that incorporation of tissue factor into the envelope of herpes simplex virus increases infection of endothelial cells and mice. Furthermore, binding of factor X to adenovirus serotype 5 enhances infection of hepatocytes but also increases the activation of the innate immune response to the virus. Coagulation proteases activate protease-activated receptors (PARs). Interestingly, we and others found that PAR1 and PAR2 modulate the immune response to viral infection. For instance, PAR1 positively regulates TLR3-dependent expression of the antiviral protein interferon β, whereas PAR2 negatively regulates expression during coxsackievirus group B infection. These studies indicate that the coagulation cascade plays multiple roles during viral infections.

  20. Preliminary crystallographic analysis of avian infectious bronchitis virus main protease

    Energy Technology Data Exchange (ETDEWEB)

    Li, Jun; Shen, Wei [Laboratory of Structural Biology, Tsinghua University, Beijing 100084 (China); Liao, Ming, E-mail: mliao@scau.edu.cn [Laboratory of Avian Medicine, College of Veterinary Medicine, South China Agricultural University, Guangzhou 510642 (China); Bartlam, Mark, E-mail: mliao@scau.edu.cn [Laboratory of Structural Biology, Tsinghua University, Beijing 100084 (China); National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101 (China)

    2007-01-01

    The avian infectious bronchitis virus main protease has been crystallized; crystals diffract to 2.7 Å resolution. Infectious bronchitis virus (IBV) is the prototype of the genus Coronavirus. It causes a highly contagious disease which affects the respiratory, reproductive, neurological and renal systems of chickens, resulting great economic losses in the poultry industry worldwide. The coronavirus (CoV) main protease (M{sup pro}), which plays a pivotal role in viral gene expression and replication through a highly complex cascade involving the proteolytic processing of replicase polyproteins, is an attractive target for antiviral drug design. In this study, IBV M{sup pro} was overexpressed in Escherichia coli. Crystals suitable for X-ray crystallography have been obtained using microseeding techniques and belong to space group P6{sub 1}22. X-ray diffraction data were collected in-house to 2.7 Å resolution from a single crystal. The unit-cell parameters were a = b = 119.1, c = 270.7 Å, α = β = 90, γ = 120°. Three molecules were predicted to be present in the asymmetric unit from a calculated self-rotation function.

  1. Heterogeneity of heat-resistant proteases from milk Pseudomonas species.

    Science.gov (United States)

    Marchand, Sophie; Vandriesche, Gonzalez; Coorevits, An; Coudijzer, Katleen; De Jonghe, Valerie; Dewettinck, Koen; De Vos, Paul; Devreese, Bart; Heyndrickx, Marc; De Block, Jan

    2009-07-31

    Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

  2. Specificity and Application of the Lantibiotic Protease NisP

    Directory of Open Access Journals (Sweden)

    Manuel Montalbán-López

    2018-02-01

    Full Text Available Lantibiotics are ribosomally produced and posttranslationally modified peptides containing several lanthionine residues. They exhibit substantial antimicrobial activity against Gram-positive bacteria, including relevant pathogens. The production of the model lantibiotic nisin minimally requires the expression of the modification and export machinery. The last step during nisin maturation is the cleavage of the leader peptide. This liberates the active compound and is catalyzed by the cell wall-anchored protease NisP. Here, we report the production and purification of a soluble variant of NisP. This has enabled us to study its specificity and test its suitability for biotechnological applications. The ability of soluble NisP to cleave leaders from various substrates was tested with two sets of nisin variants. The first set was designed to investigate the influence of amino acid variations in the leader peptide or variations around the cleavage site. The second set was designed to study the influence of the lanthionine ring topology on the proteolytic efficiency. We show that the substrate promiscuity is higher than has previously been suggested. Our results demonstrate the importance of the arginine residue at the end of the leader peptide and the importance of lanthionine rings in the substrate for specific cleavage. Collectively, these data indicate that NisP is a suitable protease for the activation of diverse heterologously expressed lantibiotics, which is required to release active antimicrobial compounds.

  3. Moringa oleifera Lam.: Protease activity against blood coagulation cascade.

    Science.gov (United States)

    Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

    2012-01-01

    The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. These findings support the traditional usage of M. oleifera extracts for wound healing.

  4. EFFECTS OF CHANGING THE INTERACTION BETWEEN SUBDOMAINS ON THE THERMOSTABILITY OF BACILLUS NEUTRAL PROTEASES

    NARCIS (Netherlands)

    EIJSINK, VGH; VRIEND, G; VANDERVINNE, B; HAZES, B; VANDENBURG, B; VENEMA, G

    1992-01-01

    Variants of the thermolabile neutral protease (Npr) of B. subtilis (Npr-sub) and the thermostable neutral protease of B. stearothermophilus (Npr-ste) were produced by means of site-directed mutagenesis and the effects of the mutations on thermostability were determined. Mutations were designed to

  5. STABILIZATION OF BACILLUS-STEAROTHERMOPHILUS NEUTRAL PROTEASE BY INTRODUCTION OF PROLINES

    NARCIS (Netherlands)

    HARDY, F; VRIEND, G; VELTMAN, OR; VANDERVINNE, B; VENEMA, G; EIJSINK, VGH

    1993-01-01

    The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by

  6. Hormone therapy affects plasma measures of factor VII-activating protease in younger postmenopausal women

    DEFF Research Database (Denmark)

    Mathiasen, Jørn Sidelmann; Skouby, S.O.; Vitzthum, F.

    2010-01-01

    Objectives Current reviews indicate that hormone therapy (HT) has a protective role in coronary heart disease (CHD) in younger postmenopausal women, whereas HT contributes to CHD in older women Factor VII-activating protease (FSAP) is a serine protease that accumulates in unstable atherosclerotic...

  7. Isolation, identification and optimization of alkaline protease production by Candida viswanathii

    Directory of Open Access Journals (Sweden)

    Mandana Lotfi

    2014-03-01

    Conclusion: Due to the high demand for industrial enzymes in the Country and the high activity of alkaline proteases produced by strain. It seems that the native strain can achieve high production of alkaline proteases.These native strains could be resulted in the independence of our country in industrial enzymes production.

  8. Mast cells limit extracellular levels of IL-13 via a serglycin proteoglycan-serine protease axis.

    Science.gov (United States)

    Waern, Ida; Karlsson, Iulia; Thorpe, Michael; Schlenner, Susan M; Feyerabend, Thorsten B; Rodewald, Hans-Reimer; Åbrink, Magnus; Hellman, Lars; Pejler, Gunnar; Wernersson, Sara

    2012-12-01

    Mast cell (MC) granules contain large amounts of proteases of the chymase, tryptase and carboxypeptidase A (MC-CPA) type that are stored in complex with serglycin,a proteoglycan with heparin side chains. Hence, serglycinprotease complexes are released upon MC degranulation and may influence local inflammation. Here we explored the possibility that a serglycin-protease axis may regulate levels of IL-13, a cytokine involved in allergic asthma. Indeed, we found that wild-type MCs efficiently degraded exogenous or endogenously produced IL-13 upon degranulation,whereas serglycin −/− MCs completely lacked this ability.Moreover, MC-mediated IL-13 degradation was blocked both by a serine protease inhibitor and by a heparin antagonist,which suggests that IL-13 degradation is catalyzed by serglycin-dependent serine proteases and that optimal IL-13 degradation is dependent on both the serglycin and the protease component of the serglycin-protease complex.Moreover, IL-13 degradation was abrogated in MC-CPA −/−MC cultures, but was normal in cultures of MCs with an inactivating mutation of MC-CPA, which suggests that the IL-13-degrading serine proteases rely on MC-CPA protein.Together, our data implicate a serglycin-serine protease axis in the regulation of extracellular levels of IL-13. Reduction of IL-13 levels through this mechanism possibly can provide a protective function in the context of allergic inflammation.

  9. Fibrinolytic protease production by new Streptomyces sp. DPUA 1576 from Amazon lichens

    Directory of Open Access Journals (Sweden)

    Germana M.M. Silva

    2015-01-01

    Conclusions: These results show that the optimization of the culture medium can enhance protease production, thus becoming a good process for further research. In addition, Streptomyces sp. DPUA 1576, isolated from Amazon lichens, might be a potential strain for fibrinolytic protease production.

  10. Protease activation involved in resistance of human cells to x-ray cell killing

    International Nuclear Information System (INIS)

    Zhang, Hong-Chang; Takahashi, Shuji; Karata, Kiyonobu; Kita, Kazuko; Suzuki, Nobuo

    2003-01-01

    Little is known of proteases that play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa-R cells after X-ray irradiation. The activity was identified as fibrinolytic, using 125 I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa-R cells showed an increased level of protease activity 10 min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with the X-ray susceptibility of cells. Leupeptin, a serine-cysteine protease inhibitor, inhibited the protease activity in samples obtained from X-ray-irradiated RSa-R cells. Treatment of RSa-R cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. However, the treatment of cells with other inhibitors tested did not modulate the X-ray susceptibility. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. (author)

  11. Lysine sulfonamides as novel HIV-protease inhibitors: Nepsilon-acyl aromatic alpha-amino acids.

    Science.gov (United States)

    Stranix, Brent R; Lavallée, Jean-François; Sévigny, Guy; Yelle, Jocelyn; Perron, Valérie; LeBerre, Nicholas; Herbart, Dominik; Wu, Jinzi J

    2006-07-01

    A series of lysine sulfonamide analogues bearing Nepsilon-acyl aromatic amino acids were synthesized using an efficient synthetic route. Evaluation of these novel protease inhibitors revealed compounds with high potency against wild-type and multiple-protease inhibitor-resistant HIV viruses.

  12. Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

    Science.gov (United States)

    Sharma, Vivek; Salwan, Richa; Sharma, Prem N

    2016-09-01

    In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

  13. Variably protease-sensitive prionopathy in the UK: a retrospective review 1991-2008

    NARCIS (Netherlands)

    Head, M.W.; Yull, H.M.; Ritchie, D.L.; Langeveld, J.P.M.; Fletcher, N.A.; Knight, R.S.; Ironside, J.W.

    2013-01-01

    Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified

  14. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group

    Energy Technology Data Exchange (ETDEWEB)

    Bungard, Christopher J.; Williams, Peter D.; Ballard, Jeanine E.; Bennett, David J.; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S.; Chang, Ronald K.; Dubost, David C.; Fay, John F.; Diamond, Tracy L.; Greshock, Thomas J.; Hao, Li; Holloway, M. Katharine; Felock, Peter J.; Gesell, Jennifer J.; Su, Hua-Poo; Manikowski, Jesse J.; McKay, Daniel J.; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M.; Nantermet, Philippe G.; Nadeau, Christian; Sanchez, Rosa I.; Satyanarayana, Tummanapalli; Shipe, William D.; Singh, Sanjay K.; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M.; Vacca, Joseph P.; Crane, Sheldon N.; McCauley, John A. (Merck); (Albany MR)

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

  15. Protease-activated receptor-1 impairs host defense in murine pneumococcal pneumonia: a controlled laboratory study

    NARCIS (Netherlands)

    Schouten, Marcel; van't Veer, Cornelis; Roelofs, Joris J. T. H.; Levi, Marcel; van der Poll, Tom

    2012-01-01

    Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor-1 (PAR-1) is expressed by multiple cell types present in the lungs and can be activated by various proteases generated during acute inflammation. The cellular effect of PAR-1

  16. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis

    Czech Academy of Sciences Publication Activity Database

    Kádek, Alan; Tretyachenko, V.; Mrázek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, M.; Schriemer, D. C.; Man, Petr

    2014-01-01

    Roč. 95, MAR 2014 (2014), s. 121-128 ISSN 1046-5928 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : Plant aspartic protease * Nepenthesin * Protease characterization Subject RIV: CE - Biochemistry Impact factor: 1.695, year: 2014

  17. Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor.

    Science.gov (United States)

    Erlandson, Martin A; Hegedus, Dwayne D; Baldwin, Douglas; Noakes, Amy; Toprak, Umut

    2010-10-01

    The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

  18. Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

    Directory of Open Access Journals (Sweden)

    Jeffrey W Koehler

    2007-02-01

    Full Text Available We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

  19. An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

    Science.gov (United States)

    Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

    2015-08-15

    A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

    Directory of Open Access Journals (Sweden)

    Pieper Rembert

    2011-05-01

    Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

  1. Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

    Science.gov (United States)

    Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H

    2015-07-01

    The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

    Science.gov (United States)

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

    2017-11-01

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

  3. Isolation and molecular characterization of cathepsin L-like cysteine protease cDNAs from Western flower thrips (Frankliniella occidentalis)

    NARCIS (Netherlands)

    Kuipers, A.G.J.; Jongsma, M.A.

    2004-01-01

    Cysteine proteases are predominant in thrips guts (TGs) and, therefore, a suitable target for selecting effective protease inhibitors against western flower thrips (Frankliniella occidentalis). We report the isolation of four full-length cysteine protease cDNA clones from thrips in a two-step PCR

  4. Expression profile of the Schistosoma japonicum degradome reveals differential protease expression patterns and potential anti-schistosomal intervention targets.

    Directory of Open Access Journals (Sweden)

    Shuai Liu

    2014-10-01

    Full Text Available Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s of individual proteases and may help to refine anti-proteolytic strategies in blood flukes.

  5. 76 FR 17843 - Intent To Compromise Claim Against the District of Columbia Public Schools

    Science.gov (United States)

    2011-03-31

    ... District of Columbia Public Schools (DCPS) now pending before the Office of Administrative Law Judges (OALJ... DEPARTMENT OF EDUCATION Intent To Compromise Claim Against the District of Columbia Public Schools... Application for Review of this PDL with the Office of Administrative Law Judges (OALJ) on September 10, 2007...

  6. Safety and Efficacy of Alginate Adhesion Barrier Gel in Compromised Intestinal Anastomosis

    NARCIS (Netherlands)

    Chaturvedi, A.; Yauw, S.T.K.; Lomme, R.M.L.M.; Hendriks, T.; Goor, H. van

    2017-01-01

    BACKGROUND: For any anti-adhesive barrier developed for abdominal surgery, the use under conditions in which anastomotic healing is compromised needs to be investigated. The current study evaluates the effect of a new ultrapure alginate gel on early healing of high-risk anastomoses in the ileum and

  7. 32 CFR 310.14 - Notification when information is lost, stolen, or compromised.

    Science.gov (United States)

    2010-07-01

    ... THE SECRETARY OF DEFENSE (CONTINUED) PRIVACY PROGRAM DOD PRIVACY PROGRAM Systems of Records § 310.14... the individual of any loss, theft, or compromise (See also, § 310.50 for reporting of the breach to Senior Component Official for Privacy and the Defense Privacy Office). (1) The notification shall be made...

  8. Towards a legitimate compromise?: An exploration of Integrated Reporting in the Netherlands

    NARCIS (Netherlands)

    van Bommel, K.

    2014-01-01

    Purpose – The purpose of this paper is to examine the multiplicity of views on integrated reporting and to consider the possibility of, and impediments to, reconciling these multiple rationales (“orders of worth”) and thus gain legitimacy through a compromise. This sheds light on the understanding

  9. Using Emergence Theory-Based Curriculum to Teach Compromise Skills to Students with Autistic Spectrum Disorders

    Science.gov (United States)

    Fein, Lance; Jones, Don

    2015-01-01

    This study addresses the compromise skills that are taught to students diagnosed with autistic spectrum disorders (ASD) and related social and communication deficits. A private school in the southeastern United States implemented an emergence theory-based curriculum to address these skills, yet no formal analysis was conducted to determine its…

  10. A method for identifying compromised clients based on DNS traffic analysis

    DEFF Research Database (Denmark)

    Stevanovic, Matija; Pedersen, Jens Myrup; D’Alconzo, Alessandro

    2017-01-01

    DNS is widely abused by Internet criminals in order to provide reliable communication within malicious network infrastructure as well as flexible and resilient hosting of malicious content. This paper presents a novel detection method that can be used for identifying potentially compromised clien...

  11. Understanding Youth's Health-Compromising Behaviors in Germany: An Application of the Risk-Behavior Framework.

    Science.gov (United States)

    Hazard, Barbara P.; Lee, Che-Fu

    1999-01-01

    Analyzed the health-compromising behaviors of German youth using responses of 2,330 seventh, eighth, and ninth graders from the German Youth Study. Smoking and drinking are not seen by these students as health-threatening behaviors, but as socially appealing behaviors. Discusses implications for health education. (SLD)

  12. Clustering of health-compromising behavior and delinquency in adolescents and adults in the Dutch population

    NARCIS (Netherlands)

    van Nieuwenhuijzen, Maroesjka; Junger, Marianne; Klein Velderman, Mariska; Wiefferink, Karin H.; Paulussen, Theo W. G. M.; Hox, Joop; Reijneveld, Sijmen A.

    Objective. This study investigates the clustering of a broad range of health-compromising and delinquent behaviors. We examine whether these behaviors belong to a single but broad cluster. 'risk-taking behavior', and whether the nature and degree of clustering in adolescents differs from that in

  13. Predicting intrapartum fetal compromise using the fetal cerebro-umbilical ratio.

    Science.gov (United States)

    Sabdia, S; Greer, R M; Prior, T; Kumar, S

    2015-05-01

    The aim of this study was to explore the association between the cerebro-umbilical ratio measured at 35-37 weeks and intrapartum fetal compromise. This retrospective cross sectional study was conducted at the Mater Mothers' Hospital in Brisbane, Australia. Maternal demographics and fetal Doppler indices at 35-37 weeks gestation for 1381 women were correlated with intrapartum and neonatal outcomes. Babies born by caesarean section or instrumental delivery for fetal compromise had the lowest median cerebro-umbilical ratio 1.60 (IQR 1.22-2.08) compared to all other delivery groups (vaginal delivery, emergency delivery for failure to progress, emergency caesarean section for other reasons or elective caesarean section). The percentage of infants with a cerebro-umbilical ratio cerebro-umbilical ratio between the 10th-90th centile and 9.6% of infants with a cerebro-umbilical ratio > 90th centile required delivery for the same indication (p cerebro-umbilical ratio was associated with an increased risk of emergency delivery for fetal compromise, OR 2.03 (95% CI 1.41-2.92), p cerebro-umbilical ratio measured at 35-37 weeks is associated with a greater risk of intrapartum compromise. This is a relatively simple technique which could be used to risk stratify women in diverse healthcare settings. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Is patient confidentiality compromised with the electronic health record?: a position paper.

    Science.gov (United States)

    Wallace, Ilse M

    2015-02-01

    In order for electronic health records to fulfill their expected benefits, protection of privacy of patient information is key. Lack of trust in confidentiality can lead to reluctance in disclosing all relevant information, which could have grave consequences. This position paper contemplates whether patient confidentiality is compromised by electronic health records. The position that confidentiality is compromised was supported by the four bioethical principles and argued that despite laws and various safeguards to protect patients' confidentiality, numerous data breaches have occurred. The position that confidentiality is not compromised was supported by virtue ethics and a utilitarian viewpoint and argued that safeguards keep information confidential and the public feels relatively safe with the electronic health record. The article concludes with an ethically superior position that confidentiality is compromised with the electronic health record. Although organizational and governmental ways of enhancing the confidentiality of patient information within the electronic health record facilitate confidentiality, the ultimate responsibility of maintaining confidentiality rests with the individual end-users and their ethical code of conduct. The American Nurses Association Code of Ethics for nurses calls for nurses to be watchful with data security in electronic communications.

  15. Advanced medical life support procedures in vitally compromised children by a helicopter emergency medical service.

    NARCIS (Netherlands)

    Gerritse, B.M.; Schalkwijk, A.; Pelzer, B.J.; Scheffer, G.J.; Draaisma, J.M.T.

    2010-01-01

    BACKGROUND: To determine the advanced life support procedures provided by an Emergency Medical Service (EMS) and a Helicopter Emergency Medical Service (HEMS) for vitally compromised children. Incidence and success rate of several procedures were studied, with a distinction made between procedures

  16. Principles of treatment planning for compromised first permanent molars in mixed dentition period: A review article

    Directory of Open Access Journals (Sweden)

    Behrad Tanbakuchi

    2017-02-01

    Full Text Available Background and Aims: The first permanent molar is susceptible to caries, endodontic complications and developmental anomalies. Compromised teeth with poor prognosis could cause the clinician to face dilemmas. The aim of this review article was to assess recent guidelines on the treatment planning of compromised first permanent molars in mixed dentition period. Materials and Methods: The design of the study was based on review of literature by searching the MEDLINE, Web of science and Google scholar. Key words (permanent first molar, extraction, treatment plan were used. This search was limited to the English articles published after the year 2000. Conclusion: A number of factors influenced decision-making process regarding compromised first permanent molars including the restorative status of the tooth, dental age of the patient, degree of crowding and occlusal relationships were assessed. The ideal time for removal of these teeth was 8-9 years of age. However, the current evidence for managing compromised first permanent molars demands clinical trials.

  17. Comment on "Compromised birth outcomes and infant mortality among racial and ethnic groups"

    NARCIS (Netherlands)

    Van der Veen, WJ

    Frisbie, Forbes, and Pullum (1996) show that it is meaningful to account for low birth weight, preterm delivery, and intrauterine growth-retardation when analyzing differences in compromised birth outcomes and infant mortality among racial and ethnic groups. I compare their findings for the 1987

  18. 7 CFR 4290.1710 - Secretary's authority to collect or compromise claims.

    Science.gov (United States)

    2010-01-01

    ... BUSINESS-COOPERATIVE SERVICE AND RURAL UTILITIES SERVICE, DEPARTMENT OF AGRICULTURE RURAL BUSINESS INVESTMENT COMPANY (âRBICâ) PROGRAM Financial Assistance for RBICs (Leverage) Miscellaneous § 4290.1710... consideration as he or she deems reasonable, collect or compromise all claims relating to obligations he or she...

  19. Rumen microbial genomics

    International Nuclear Information System (INIS)

    Morrison, M.; Nelson, K.E.

    2005-01-01

    Improving microbial degradation of plant cell wall polysaccharides remains one of the highest priority goals for all livestock enterprises, including the cattle herds and draught animals of developing countries. The North American Consortium for Genomics of Fibrolytic Ruminal Bacteria was created to promote the sequencing and comparative analysis of rumen microbial genomes, offering the potential to fully assess the genetic potential in a functional and comparative fashion. It has been found that the Fibrobacter succinogenes genome encodes many more endoglucanases and cellodextrinases than previously isolated, and several new processive endoglucanases have been identified by genome and proteomic analysis of Ruminococcus albus, in addition to a variety of strategies for its adhesion to fibre. The ramifications of acquiring genome sequence data for rumen microorganisms are profound, including the potential to elucidate and overcome the biochemical, ecological or physiological processes that are rate limiting for ruminal fibre degradation. (author)

  20. Microbial Genomes Multiply

    Science.gov (United States)

    Doolittle, Russell F.

    2002-01-01

    The publication of the first complete sequence of a bacterial genome in 1995 was a signal event, underscored by the fact that the article has been cited more than 2,100 times during the intervening seven years. It was a marvelous technical achievement, made possible by automatic DNA-sequencing machines. The feat is the more impressive in that complete genome sequencing has now been adopted in many different laboratories around the world. Four years ago in these columns I examined the situation after a dozen microbial genomes had been completed. Now, with upwards of 60 microbial genome sequences determined and twice that many in progress, it seems reasonable to assess just what is being learned. Are new concepts emerging about how cells work? Have there been practical benefits in the fields of medicine and agriculture? Is it feasible to determine the genomic sequence of every bacterial species on Earth? The answers to these questions maybe Yes, Perhaps, and No, respectively.