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Sample records for 3c protease complexed

  1. Poliovirus protease 3C(pro) kills cells by apoptosis.

    Science.gov (United States)

    Barco, A; Feduchi, E; Carrasco, L

    2000-01-20

    The tetracycline-based Tet-Off expression system has been used to analyze the effects of poliovirus protease 3C(pro) on human cells. Stable HeLa cell clones that express this poliovirus protease under the control of an inducible, tightly regulated promoter were obtained. Tetracycline removal induces synthesis of 3C protease, followed by drastic morphological alterations and cellular death. Degradation of cellular DNA in nucleosomes and generation of apoptotic bodies are observed from the second day after 3C(pro) induction. The cleavage of poly(ADP-ribose) polymerase, an enzyme involved in DNA repair, occurs after induction of 3C(pro), indicating caspase activation by this poliovirus protease. The 3C(pro)-induced apoptosis is blocked by the caspase inhibitor z-VAD-fmk. Our findings suggest that the protease 3C is responsible for triggering apoptosis in poliovirus-infected cells by a mechanism that involves caspase activation.

  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

    2013-02-01

    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 a feline coronavirus in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC(50) in a nanomolar range) and, furthermore, combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in a cell culture system.

  3. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    OpenAIRE

    Raheem Ullah; Majid Ali Shah; Soban Tufail; Fouzia Ismat; Muhammad Imran; Mazhar Iqbal; Osman Mirza; Moazur Rhaman

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally ...

  4. Cleavage of Maize chlorotic dwarf virus R78 protein by the viral 3C protease

    Science.gov (United States)

    Maize chlorotic dwarf virus (MCDV) is a member of the genus Waikavirus and encodes a 389 kDa polyprotein from its 11784 nt genomic RNA. Like many polyprotein-encoding viruses, MCDV contains a 3C-like virus protease that is presumably responsible for maturation cleavages of the polyprotein. However,...

  5. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Directory of Open Access Journals (Sweden)

    Raheem Ullah

    Full Text Available Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  6. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Science.gov (United States)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications. PMID:27093053

  7. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production.

    Science.gov (United States)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban; Ismat, Fouzia; Imran, Muhammad; Iqbal, Mazhar; Mirza, Osman; Rhaman, Moazur

    2016-01-01

    Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three different fusion proteins at 4°C, a temperature of choice for purification of many proteins. The results show that the human rhinovirus 3C protease performs better at 4°C than the frequently used tobacco etch virus protease and its activity was insensitive to most of the experimental conditions tested. Though number of fusion proteins tested is limited, we expect that these finding will facilitate the use of the human rhinovirus 3C protease in recombinant protein production for pharmaceutical and biotechnological applications.

  8. Activity of the Human Rhinovirus 3C Protease Studied in Various Buffers, Additives and Detergents Solutions for Recombinant Protein Production

    DEFF Research Database (Denmark)

    Ullah, Raheem; Shah, Majid Ali; Tufail, Soban;

    2016-01-01

    and time-consuming procedures before proteolysis can occur. To address this issue, we examined the effect of elution buffers used for common affinity based purifications, salt ions, stability/solubility and reducing agents, and detergents on the activity of the human rhinovirus 3C protease using three......Proteases are widely used to remove affinity and solubility tags from recombinant proteins to avoid potential interference of these tags with the structure and function of the fusion partner. In recent years, great interest has been seen in use of the human rhinovirus 3C protease owing to its...

  9. Design and structure-activity relationships of novel inhibitors of human rhinovirus 3C protease.

    Science.gov (United States)

    Kawatkar, S P; Gagnon, M; Hoesch, V; Tiong-Yip, C; Johnson, K; Ek, M; Nilsson, E; Lister, T; Olsson, L; Patel, J; Yu, Q

    2016-07-15

    Human rhinovirus (HRV) is a primary cause of common cold and is linked to exacerbation of underlying respiratory diseases such as asthma and COPD. HRV 3C protease, which is responsible for cleavage of viral polyprotein in to proteins essential for viral life-cycle, represents an important target. We have designed proline- and azetidine-based analogues of Rupintrivir that target the P2 pocket of the binding site. Potency optimization, aided with X-ray crystallography and quantum mechanical calculations, led to compounds with activity against a broad spectrum of HRV serotypes. Altogether, these compounds represent alternative starting points to identify promising leads in our continual efforts to treat HRV infections. PMID:27265257

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

    Directory of Open Access Journals (Sweden)

    Erin J Walker

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

  11. Crystal structure of the 3C protease from Southern African Territories type 2 foot-and-mouth disease virus.

    Science.gov (United States)

    Yang, Jingjie; Leen, Eoin N; Maree, Francois F; Curry, Stephen

    2016-01-01

    The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3C(pro)). As in other picornaviruses, 3C(pro) performs most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3C(pro) from serotype A-one of the seven serotypes of FMDV-adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3C(pro) amino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3C(pro) from SAT2/GHA/8/91. PMID:27168976

  12. Enterovirus 71 3C protease cleaves a novel target CstF-64 and inhibits cellular polyadenylation.

    Directory of Open Access Journals (Sweden)

    Kuo-Feng Weng

    2009-09-01

    Full Text Available Identification of novel cellular proteins as substrates to viral proteases would provide a new insight into the mechanism of cell-virus interplay. Eight nuclear proteins as potential targets for enterovirus 71 (EV71 3C protease (3C(pro cleavages were identified by 2D electrophoresis and MALDI-TOF analysis. Of these proteins, CstF-64, which is a critical factor for 3' pre-mRNA processing in a cell nucleus, was selected for further study. A time-course study to monitor the expression levels of CstF-64 in EV71-infected cells also revealed that the reduction of CstF-64 during virus infection was correlated with the production of viral 3C(pro. CstF-64 was cleaved in vitro by 3C(pro but neither by mutant 3C(pro (in which the catalytic site was inactivated nor by another EV71 protease 2A(pro. Serial mutagenesis was performed in CstF-64, revealing that the 3C(pro cleavage sites are located at position 251 in the N-terminal P/G-rich domain and at multiple positions close to the C-terminus of CstF-64 (around position 500. An accumulation of unprocessed pre-mRNA and the depression of mature mRNA were observed in EV71-infected cells. An in vitro assay revealed the inhibition of the 3'-end pre-mRNA processing and polyadenylation in 3C(pro-treated nuclear extract, and this impairment was rescued by adding purified recombinant CstF-64 protein. In summing up the above results, we suggest that 3C(pro cleavage inactivates CstF-64 and impairs the host cell polyadenylation in vitro, as well as in virus-infected cells. This finding is, to our knowledge, the first to demonstrate that a picornavirus protein affects the polyadenylation of host mRNA.

  13. IRES mediated expression of viral 3C protease for enhancing the yield of FMDV empty capsids using baculovirus system.

    Science.gov (United States)

    Vivek Srinivas, V M; Basagoudanavar, Suresh H; Hosamani, Madhusudan

    2016-03-01

    For expression of FMDV empty capsids, high protease activity associated with 3C co-expressed with P1 polyprotein has been reported to adversely affect the yields of capsids. Limiting the levels of 3Cpro relative to P1-2A polypeptide is thus critical to enhance the yields. In this study, FMDV internal ribosome entry site (IRES) sequence which serves as an alternative to the CAP-dependent translation initiation mechanism, was used for controlled translation of 3C protease. Baculovirus expressing bicistronic cDNA cassette containing two open reading frames-FMDV capsid gene (P1-2A) and 3Cpro intervened by IRES was prepared. Analysis of the expression in insect cells infected with baculovirus showed increased accumulation of processed capsids. Recombinant capsids showed higher immunoreactivity similar to the whole virus antigen, when reacted with polyclonal antibodies against the purified whole virus 146S particles. Thus, inclusion of the IRES upstream of 3Cpro facilitated reduced expression of the protease in baculovirus expression system, without causing significant proteolysis, thereby contributing to improved yields of the processed capsid antigens. PMID:26775685

  14. Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease

    Science.gov (United States)

    Tan, Yong Wah; Ang, Melgious Jin Yan; Lau, Qiu Ying; Poulsen, Anders; Ng, Fui Mee; Then, Siew Wen; Peng, Jianhe; Hill, Jeffrey; Hong, Wan Jin; Chia, Cheng San Brian; Chu, Justin Jang Hann

    2016-01-01

    Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery. PMID:27645381

  15. Antiviral activities of peptide-based covalent inhibitors of the Enterovirus 71 3C protease.

    Science.gov (United States)

    Tan, Yong Wah; Ang, Melgious Jin Yan; Lau, Qiu Ying; Poulsen, Anders; Ng, Fui Mee; Then, Siew Wen; Peng, Jianhe; Hill, Jeffrey; Hong, Wan Jin; Chia, Cheng San Brian; Chu, Justin Jang Hann

    2016-01-01

    Hand, Foot and Mouth Disease is a highly contagious disease caused by a range of human enteroviruses. Outbreaks occur regularly, especially in the Asia-Pacific region, putting a burden on public healthcare systems. Currently, there is no antiviral for treating this infectious disease and the only vaccines are limited to circulation in China, presenting an unmet medical need that needs to be filled urgently. The human enterovirus 3 C protease has been deemed a plausible drug target due to its essential roles in viral replication. In this study, we designed and synthesized 10 analogues of the Rhinovirus 3 C protease inhibitor, Rupintrivir, and tested their 3 C protease inhibitory activities followed by a cellular assay using human enterovirus 71 (EV71)-infected human RD cells. Our results revealed that a peptide-based compound containing a trifluoromethyl moiety to be the most potent analogue, with an EC50 of 65 nM, suggesting its potential as a lead for antiviral drug discovery. PMID:27645381

  16. X-ray structure at 1.75 resolution of a norovirus 3C protease linked to an active site-directed peptide inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Cooper, Jon [University of Southampton, England; Coates, Leighton [ORNL; Hussey, Robert [University of Southampton, England

    2010-01-01

    Noroviruses are recognized universally as the most important cause of human epidemic non-bacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.75 resolution, following initial MAD phasing with a selenomethionine derivative. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, based on a 3C protease cleavage recognition sequences in the 200kDa polyprotein substrate, reacts covalently through its propenylethylester group (X) with the active site nucleophile, Cys 139. The 3C protease-inhibitor structure permits, for the first time, the identification of substrate recognition and binding groups and provides important new information for the development of antiviral prophylactics.

  17. Crystal structure of the 3C protease from Southern African Territories type 2 foot-and-mouth disease virus

    Science.gov (United States)

    Yang, Jingjie; Leen, Eoin N.; Maree, Francois F.

    2016-01-01

    The replication of foot-and-mouth disease virus (FMDV) is dependent on the virus-encoded 3C protease (3Cpro). As in other picornaviruses, 3Cpro performs most of the proteolytic processing of the polyprotein expressed from the large open reading frame in the RNA genome of the virus. Previous work revealed that the 3Cpro from serotype A—one of the seven serotypes of FMDV—adopts a trypsin-like fold. On the basis of capsid sequence comparisons the FMDV serotypes are grouped into two phylogenetic clusters, with O, A, C, and Asia 1 in one, and the three Southern African Territories serotypes, (SAT-1, SAT-2 and SAT-3) in another, a grouping pattern that is broadly, but not rigidly, reflected in 3Cpro amino acid sequences. We report here the cloning, expression and purification of 3C proteases from four SAT serotype viruses (SAT2/GHA/8/91, SAT1/NIG/5/81, SAT1/UGA/1/97, and SAT2/ZIM/7/83) and the crystal structure at 3.2 Å resolution of 3Cpro from SAT2/GHA/8/91. PMID:27168976

  18. A mammalian cell-based reverse two-hybrid system for functional analysis of 3C viral protease of human enterovirus 71.

    Science.gov (United States)

    Lee, Jin-Ching; Shih, Shin-Ru; Chang, Ten-Yuan; Tseng, Huan-Yi; Shih, Ya-Feng; Yen, Kuei-Jung; Chen, Wei-Chun; Shie, Jiun-Jie; Fang, Jim-Min; Liang, Po-Huang; Chao, Yu-Sheng; Hsu, John T-A

    2008-04-01

    Although several cell-based reporter assays have been developed for screening of viral protease inhibitors, most of these assays have a significant limitation in that numerous false positives can be generated for the compounds that are interfering with reporter gene detection due to the cellular viability. To improve, we developed a mammalian cell-based assay based on the reverse two-hybrid system to monitor the proteolytic activity of human enterovirus 71 (EV71) 3C protease and to validate the cytotoxicity of compounds at the same time. In this system, the GAL4 DNA binding domain (M3) and transactivation domain (VP16) were fused, in-frame, with 3C or 3C(mut). The 3C(mut) was an inactivated protease with mutations at the predicted catalytic triad. The reporter plasmid contains a secreted alkaline phosphatase (SEAP) gene under the control of GAL4 activating sequences. We demonstrated that M3-3C-VP16 failed to turn on the expression of SEAP due to the separation of M3 and the VP16 domains by self-cleavage of 3C. In contrast, SEAP expression was induced by the M3-3C(mut)-VP16 fusion protein or the M3-3C-VP16 in cells treated with AG7088, a potent inhibitor of human rhinoviruses (HRVs) 3C protease. Potentially, this protease detection system should greatly facilitate anti-EV71 drug discovery through a high-throughput screening. PMID:18190777

  19. Enzymatic activity characterization of SARS coronavirus 3C-like protease by fluorescence resonance energy transfer technique

    Institute of Scientific and Technical Information of China (English)

    Shuai CHEN; Hua-liang JIANG; Li-li CHEN; Hai-bin LUO; Tao SUN; Jing CHEN; Fei YE; Jian-hua CAI; Jing-kang SHEN; Xu SHEN

    2005-01-01

    Aim: To characterize enzymatic activity of severe acute respiratory syndrome(SARS) coronavirus (CoV) 3C-like protease (3CLpro) and its four site-directed mutants. Methods: Based on the fluorescence resonance energy transfer (FRET)principle using 5-[(2'-aminoethyl)-amino] naphthelenesulfonic acid (EDANS) and 4-[[4-(dimethylamino) phenyl] azo] benzoic acid (Dabcyl) as the energy transfer pair, one fluorogenic substrate was designed for the evaluation of SARS-CoV 3CLpro proteolytic activity. Results: The kinetic parameters of the fluorogenic substrate have been determined as Km=404 μmol.L-1, kcat=1.08 min-1, and kcat/Km=2.7 gered activity switches, and site-directed mutagenesis analysis of SARS-CoV 3CLpro revealed that substitutions of His41, Cys145, and His163 resulted in complete loss of enzymatic activity, while replacement of Met162 with Ala caused strongly increased activity. Conclusion: This present work has provided valuable information for understanding the catalytic mechanism of SARS-CoV 3CLpro. This FRET-based assay might supply an ideal approach for the exploration SARSCoV 3CLpro putative inhibitors.

  20. Inhibition of SARS-CoV 3C-like Protease Activity by Theaflavin-3,3'-digallate (TF3

    Directory of Open Access Journals (Sweden)

    Chia-Nan Chen

    2005-01-01

    Full Text Available SARS-CoV is the causative agent of severe acute respiratory syndrome (SARS. The virally encoded 3C-like protease (3CLPro has been presumed critical for the viral replication of SARS-CoV in infected host cells. In this study, we screened a natural product library consisting of 720 compounds for inhibitory activity against 3CLPro. Two compounds in the library were found to be inhibitive: tannic acid (IC50 = 3 µM and 3-isotheaflavin-3-gallate (TF2B (IC50 = 7 µM. These two compounds belong to a group of natural polyphenols found in tea. We further investigated the 3CLPro-inhibitory activity of extracts from several different types of teas, including green tea, oolong tea, Puer tea and black tea. Our results indicated that extracts from Puer and black tea were more potent than that from green or oolong teas in their inhibitory activities against 3CLPro. Several other known compositions in teas were also evaluated for their activities in inhibiting 3CLPro. We found that caffeine, (—-epigallocatechin gallte (EGCg, epicatechin (EC, theophylline (TP, catechin (C, epicatechin gallate (ECg and epigallocatechin (EGC did not inhibit 3CLPro activity. Only theaflavin-3,3′-digallate (TF3 was found to be a 3CLPro inhibitor. This study has resulted in the identification of new compounds that are effective 3CLPro inhibitors.

  1. Low levels of foot-and-mouth disease virus 3C protease expression are required to achieve optimal capsid protein expression and processing in mammalian cells

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Gullberg, Maria; Li, Jiong;

    2013-01-01

    The foot-and-mouth disease virus (FMDV) capsid protein precursor (P1-2A) is processed by the virus-encoded 3C protease (3Cpro) to produce VP0, VP3, VP1 and 2A. Within the virus-encoded polyprotein, the P1-2A and 3Cpro can be expected to be produced at equivalent concentrations. However, using...... production of diagnostic reagents and improved vaccines against foot-and-mouth disease....

  2. Dynamically-driven inactivation of the catalytic machinery of the SARS 3C-like protease by the N214A mutation on the extra domain.

    Science.gov (United States)

    Shi, Jiahai; Han, Nanyu; Lim, Liangzhong; Lua, Shixiong; Sivaraman, J; Wang, Lushan; Mu, Yuguang; Song, Jianxing

    2011-02-01

    Despite utilizing the same chymotrypsin fold to host the catalytic machinery, coronavirus 3C-like proteases (3CLpro) noticeably differ from picornavirus 3C proteases in acquiring an extra helical domain in evolution. Previously, the extra domain was demonstrated to regulate the catalysis of the SARS-CoV 3CLpro by controlling its dimerization. Here, we studied N214A, another mutant with only a doubled dissociation constant but significantly abolished activity. Unexpectedly, N214A still adopts the dimeric structure almost identical to that of the wild-type (WT) enzyme. Thus, we conducted 30-ns molecular dynamics (MD) simulations for N214A, WT, and R298A which we previously characterized to be a monomer with the collapsed catalytic machinery. Remarkably, three proteases display distinctive dynamical behaviors. While in WT, the catalytic machinery stably retains in the activated state; in R298A it remains largely collapsed in the inactivated state, thus implying that two states are not only structurally very distinguishable but also dynamically well separated. Surprisingly, in N214A the catalytic dyad becomes dynamically unstable and many residues constituting the catalytic machinery jump to sample the conformations highly resembling those of R298A. Therefore, the N214A mutation appears to trigger the dramatic change of the enzyme dynamics in the context of the dimeric form which ultimately inactivates the catalytic machinery. The present MD simulations represent the longest reported so far for the SARS-CoV 3CLpro, unveiling that its catalysis is critically dependent on the dynamics, which can be amazingly modulated by the extra domain. Consequently, mediating the dynamics may offer a potential avenue to inhibit the SARS-CoV 3CLpro.

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

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    , introduction of the 2A L2P substitution alone, or with the VP1 K210E change, into this virus resulted in the production of viable viruses. Cells infected with viruses containing the VP1 K210E and/or the 2A L2P substitutions contained the uncleaved VP1-2A protein; the 2A L2P substitution rendered the VP1/2A......The foot-and-mouth disease virus (FMDV) capsid protein precursor P1-2A is cleaved by the 3C protease to produce VP0, VP3, VP1 and 2A. It was shown previously that modification of a single amino acid residue (K210) within the VP1 protein, close to the VP1/2A cleavage site, inhibited cleavage...... of this junction and resulted in the production of “self-tagged” virus particles containing the 2A peptide. A second site substitution (E83K) within VP1 was also observed within the rescued virus (Gullberg et al., 2013). It is now shown that introduction of this E83K change alone into a serotype O virus resulted...

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

    DEFF Research Database (Denmark)

    Gullberg, Maria; Polacek, Charlotta; Belsham, Graham

    2014-01-01

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

  5. GA binding protein augments autophagy via transcriptional activation of BECN1-PIK3C3 complex genes.

    Science.gov (United States)

    Zhu, Wan; Swaminathan, Gayathri; Plowey, Edward D

    2014-09-01

    Macroautophagy is a vesicular catabolic trafficking pathway that is thought to protect cells from diverse stressors and to promote longevity. Recent studies have revealed that transcription factors play important roles in the regulation of autophagy. In this study, we have identified GA binding protein (GABP) as a transcriptional regulator of the combinatorial expression of BECN1-PIK3C3 complex genes involved in autophagosome initiation. We performed bioinformatics analyses that demonstrated highly conserved putative GABP sites in genes that encode BECN1/Beclin 1, several BECN1 interacting proteins, and downstream autophagy proteins including the ATG12-ATG5-ATG16L1 complex. We demonstrate that GABP binds to the promoter regions of BECN1-PIK3C3 complex genes and activates their transcriptional activities. Knockdown of GABP reduced BECN1-PIK3C3 complex transcripts, BECN1-PIK3C3 complex protein levels and autophagy in cultured cells. Conversely, overexpression of GABP increased autophagy. Nutrient starvation increased GABP-dependent transcriptional activity of BECN1-PIK3C3 complex gene promoters and increased the recruitment of GABP to the BECN1 promoter. Our data reveal a novel function of GABP in the regulation of autophagy via transcriptional activation of the BECN1-PIK3C3 complex.

  6. Shutoff of RNA polymerase II transcription by poliovirus involves 3C protease-mediated cleavage of the TATA-binding protein at an alternative site: incomplete shutoff of transcription interferes with efficient viral replication.

    Science.gov (United States)

    Kundu, Pallob; Raychaudhuri, Santanu; Tsai, Weimin; Dasgupta, Asim

    2005-08-01

    The TATA-binding protein (TBP) plays a crucial role in cellular transcription catalyzed by all three DNA-dependent RNA polymerases. Previous studies have shown that TBP is targeted by the poliovirus (PV)-encoded protease 3C(pro) to bring about shutoff of cellular RNA polymerase II-mediated transcription in PV-infected cells. The processing of the majority of viral precursor proteins by 3C(pro) involves cleavages at glutamine-glycine (Q-G) sites. We present evidence that suggests that the transcriptional inactivation of TBP by 3C(pro) involves cleavage at the glutamine 104-serine 105 (Q104-S105) site of TBP and not at the Q18-G19 site as previously thought. The TBP Q104-S105 cleavage by 3C(pro) is greatly influenced by the presence of an aliphatic amino acid at the P4 position, a hallmark of 3C(pro)-mediated proteolysis. To examine the importance of host cell transcription shutoff in the PV life cycle, stable HeLa cell lines were created that express recombinant TBP resistant to cleavage by the viral proteases, called GG rTBP. Transcription shutoff was significantly impaired and delayed in GG rTBP cells upon infection with poliovirus compared with the cells that express wild-type recombinant TBP (wt rTBP). Infection of GG rTBP cells with poliovirus resulted in small plaques, significantly reduced viral RNA synthesis, and lower viral yields compared to the wt rTBP cell line. These results suggest that a defect in transcription shutoff can lead to inefficient replication of poliovirus in cultured cells.

  7. Aspects of preanalytical variation of lactoferrin and elastase/alpha 1-protease inhibitor complexes

    DEFF Research Database (Denmark)

    Antonsen, S; Qvist, N; Wanscher, M

    1993-01-01

    A number of interesting applications of plasma elastase/alpha 1-protease inhibitor complexes (ELA-PI) and lactoferrin (LAC) have recently been suggested. However, the clinical utility of these components often seems to be low. This might be improved by minimizing the preanalytical variation...

  8. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    Energy Technology Data Exchange (ETDEWEB)

    Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth, E-mail: elizabeth.rieder@ars.usda.gov

    2012-03-30

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3C{sup pro} induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5 Prime non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3C{sup pro}.

  9. Structure of a murine norovirus NS6 protease-product complex revealed by adventitious crystallisation.

    Directory of Open Access Journals (Sweden)

    Eoin N Leen

    Full Text Available Murine noroviruses have emerged as a valuable tool for investigating the molecular basis of infection and pathogenesis of the closely related human noroviruses, which are the major cause of non-bacterial gastroenteritis. The replication of noroviruses relies on the proteolytic processing of a large polyprotein precursor into six non-structural proteins (NS1-2, NS3, NS4, NS5, NS6(pro, NS7(pol by the virally-encoded NS6 protease. We report here the crystal structure of MNV NS6(pro, which has been determined to a resolution of 1.6 Å. Adventitiously, the crystal contacts are mediated in part by the binding of the C-terminus of NS6(pro within the peptide-binding cleft of a neighbouring molecule. This insertion occurs for both molecules in the asymmetric unit of the crystal in a manner that is consistent with physiologically-relevant binding, thereby providing two independent views of a protease-peptide complex. Since the NS6(pro C-terminus is formed in vivo by NS6(pro processing, these crystal contacts replicate the protease-product complex that is formed immediately following cleavage of the peptide bond at the NS6-NS7 junction. The observed mode of binding of the C-terminal product peptide yields new insights into the structural basis of NS6(pro specificity.

  10. Serotype-Specific Structural Differences in the Protease-Cofactor Complexes of the Dengue Virus Family

    Energy Technology Data Exchange (ETDEWEB)

    Chandramouli, Sumana; Joseph, Jeremiah S.; Daudenarde, Sophie; Gatchalian, Jovylyn; Cornillez-Ty, Cromwell; Kuhn, Peter (Scripps)

    2010-03-04

    With an estimated 40% of the world population at risk, dengue poses a significant threat to human health, especially in tropical and subtropical regions. Preventative and curative efforts, such as vaccine development and drug discovery, face additional challenges due to the occurrence of four antigenically distinct serotypes of the causative dengue virus (DEN1 to -4). Complex immune responses resulting from repeat assaults by the different serotypes necessitate simultaneous targeting of all forms of the virus. One of the promising targets for drug development is the highly conserved two-component viral protease NS2B-NS3, which plays an essential role in viral replication by processing the viral precursor polyprotein into functional proteins. In this paper, we report the 2.1-{angstrom} crystal structure of the DEN1 NS2B hydrophilic core (residues 49 to 95) in complex with the NS3 protease domain (residues 1 to 186) carrying an internal deletion in the N terminus (residues 11 to 20). While the overall folds within the protease core are similar to those of DEN2 and DEN4 proteases, the conformation of the cofactor NS2B is dramatically different from those of other flaviviral apoprotease structures. The differences are especially apparent within its C-terminal region, implicated in substrate binding. The structure reveals for the first time serotype-specific structural elements in the dengue virus family, with the reported alternate conformation resulting from a unique metal-binding site within the DEN1 sequence. We also report the identification of a 10-residue stretch within NS3pro that separates the substrate-binding function from the catalytic turnover rate of the enzyme. Implications for broad-spectrum drug discovery are discussed.

  11. X-ray structure and inhibition of the feline infectious peritonitis virus 3C-like protease: Structural implications for drug design.

    Science.gov (United States)

    St John, Sarah E; Therkelsen, Matthew D; Nyalapatla, Prasanth R; Osswald, Heather L; Ghosh, Arun K; Mesecar, Andrew D

    2015-11-15

    Feline infectious peritonitis (FIP) is a deadly disease that effects both domestic and wild cats and is caused by a mutation in feline coronavirus (FCoV) that allows the virus to replicate in macrophages. Currently, there are no treatments or vaccines available for the treatment of FIP even though it kills approximately 5% of cats in multi-cat households per year. In an effort to develop small molecule drugs targeting FIP for the treatment of cats, we screened a small set of designed peptidomimetic inhibitors for inhibition of FIPV-3CL(pro), identifying two compounds with low to sub-micromolar inhibition, compound 6 (IC50=0.59±0.06 μM) and compound 7 (IC50=1.3±0.1 μM). We determined the first X-ray crystal structure of FIPV-3CL(pro) in complex with the best inhibitor identified, compound 6, to a resolution of 2.10 Å to better understand the structural basis for inhibitor specificity. Our study provides important insights into the structural requirements for the inhibition of FIPV-3CL(pro) by peptidomimetic inhibitors and expands the current structural knowledge of coronaviral 3CL(pro) architecture.

  12. Crystal structure of Zika virus NS2B-NS3 protease in complex with a boronate inhibitor.

    Science.gov (United States)

    Lei, Jian; Hansen, Guido; Nitsche, Christoph; Klein, Christian D; Zhang, Linlin; Hilgenfeld, Rolf

    2016-07-29

    The ongoing Zika virus (ZIKV) outbreak is linked to severe neurological disorders. ZIKV relies on its NS2B/NS3 protease for polyprotein processing; hence, this enzyme is an attractive drug target. The 2.7 angstrom; crystal structure of ZIKV protease in complex with a peptidomimetic boronic acid inhibitor reveals a cyclic diester between the boronic acid and glycerol. The P2 4-aminomethylphenylalanine moiety of the inhibitor forms a salt-bridge with the nonconserved Asp(83) of NS2B; ion-pairing between Asp(83) and the P2 residue of the substrate likely accounts for the enzyme's high catalytic efficiency. The unusual dimer of the ZIKV protease:inhibitor complex seen in the crystal may provide a model for assemblies formed at high local concentrations of protease at the endoplasmatic reticulum membrane, the site of polyprotein processing. PMID:27386922

  13. The complex circumnuclear environment of the broad-line radio galaxy 3C 390.3 revealed by Chandra HETG

    CERN Document Server

    Tombesi, F; Kallman, T; Reynolds, C S; Mushotzky, R F; Braito, V; Behar, E; Leutenegger, M A; Cappi, M

    2016-01-01

    We present the first high spectral resolution X-ray observation of the broad-line radio galaxy 3C 390.3 obtained with the high energy transmission grating (HETG) spectrometer on board the Chandra X-ray Observatory. The spectrum shows complex emission and absorption features in both the soft X-rays and Fe K band. We detect emission and absorption lines in the energy range between E = 700-1000 eV associated with ionized Fe L transitions (Fe XVII-XX). An emission line at the energy of E=6.4 keV consistent with the Fe K\\alpha is also observed. Our best-fit model requires at least three different components: (i) a hot emission component likely associated with the hot interstellar medium in this elliptical galaxy with temperature kT=0.5+/-0.1 keV; (ii) a warm absorber with ionization parameter log\\xi=2.3+/-0.5 erg s^{-1} cm, column density logN_H=20.7+/-0.1 cm^{-2}, and outflow velocity of v_{out}<150 km s^{-1}; (iii) a lowly ionized reflection component in the Fe K band likely associated with the optical broad ...

  14. Effects of nanosuspension and inclusion complex techniques on the in vitro protease inhibitory activity of naproxen

    Energy Technology Data Exchange (ETDEWEB)

    Dharmalingam, Senthil Rajan; Chidambaram, Kumarappan; Srinivasan, Ramamurthy; Nadaraju, Shamala, E-mail: dsenthilrajan@yahoo.co.in [School of Pharmacy, International Medical University, Bukit Jalil, Kuala Lumpur (Malaysia)

    2014-01-15

    This study investigated the effects of nanosuspension and inclusion complex techniques on in vitro trypsin inhibitory activity of naproxen—a member of the propionic acid derivatives, which are a group of antipyretic, analgesic, and non-steroidal anti-inflammatory drugs. Nanosuspension and inclusion complex techniques were used to increase the solubility and anti-inflammatory efficacy of naproxen. The evaporative precipitation into aqueous solution (EPAS) technique and the kneading methods were used to prepare the nanosuspension and inclusion complex of naproxen, respectively. We also used an in vitro protease inhibitory assay to investigate the anti-inflammatory effect of modified naproxen formulations. Physiochemical properties of modified naproxen formulations were analyzed using UV, IR spectra, and solubility studies. Beta-cyclodextrin inclusion complex of naproxen was found to have a lower percentage of antitryptic activity than a pure nanosuspension of naproxen did. In conclusion, nanosuspension of naproxen has a greater anti-inflammatory effect than the other two tested formulations. This is because the nanosuspension formulation reduces the particle size of naproxen. Based on these results, the antitryptic activity of naproxen nanosuspension was noteworthy; therefore, this formulation can be used for the management of inflammatory disorders. (author)

  15. NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir

    Energy Technology Data Exchange (ETDEWEB)

    Furukawa, Ayako; Okamura, Hideyasu [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Morishita, Ryo [CellFree Sciences Co. Ltd., Ehime University, Venture Business Laboratory, Matsuyama 790-8577 (Japan); Department of Microbiology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004 (Japan); Matsunaga, Satoko [Department of Microbiology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004 (Japan); Kobayashi, Naohiro; Ikegami, Takahisa [Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-0871 (Japan); Kodaki, Tsutomu [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Graduate School of Energy Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Takaori-Kondo, Akifumi [Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507 (Japan); Ryo, Akihide [Department of Microbiology, Yokohama City University School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama 236-0004 (Japan); Nagata, Takashi, E-mail: nagatat@iae.kyoto-u.ac.jp [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Graduate School of Energy Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Katahira, Masato, E-mail: katahira@iae.kyoto-u.ac.jp [Institute of Advanced Energy, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); Graduate School of Energy Science, Kyoto University, Gokasho, Uji, Kyoto 611-0011 (Japan); CREST, JST, 5-3 Yonban-cho, Chiyoda-ku, Tokyo 102-8666 (Japan)

    2012-08-24

    Highlights: Black-Right-Pointing-Pointer Protease (PR) of XMR virus (XMRV) was successfully synthesized with cell-free system. Black-Right-Pointing-Pointer Interface of XMRV PR with an inhibitor, amprenavir (APV), was identified with NMR. Black-Right-Pointing-Pointer Structural heterogeneity is induced for two PR protomers in the APV:PR = 1:2 complex. Black-Right-Pointing-Pointer Structural heterogeneity is transmitted even to distant regions from the interface. Black-Right-Pointing-Pointer Long-range transmission of structural change may be utilized for drug discovery. -- Abstract: Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially {sup 15}N-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [{sup 1}H, {sup 15}N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) . A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR = 1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR

  16. Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

    NARCIS (Netherlands)

    Janssen, B.J.C.; Halff, E.F.; Lambris, J.D.; Gros, P.

    2007-01-01

    Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step

  17. Structure of Compstatin in Complex with Complement Component C3c Reveals a New Mechanism of Complement Inhibition

    OpenAIRE

    Janssen, B.J.C.; Halff, E.F.; LAMBRIS, J. D.; Gros, P

    2007-01-01

    Undesired complement activation is a major cause of tissue injury in various pathological conditions and contributes to several immune complex diseases. Compstatin, a 13-residue peptide, is an effective inhibitor of the activation of complement component C3 and thus blocks a central and crucial step in the complement cascade. The precise binding site on C3, the structure in the bound form, and the exact mode of action of compstatin are unknown. Here we present the crystal structure of compsta...

  18. Dynamical analysis of the complex radio structure in 3C 293: Clues on a rapid jet realignment in X-shaped radio galaxies

    CERN Document Server

    Machalski, J; Stawarz, L; Wezgowiec, M

    2016-01-01

    Radio galaxies classified as X-shaped/winged, are characterised by two pairs of extended and misaligned lobes, which suggest a rapid realignment of the jet axis, for which a potential cause is still under debate. Here we analyse the complex radio structure of 3C 293 winged source hosted by the post-merger galaxy, which uniquely displays a significant asymmetry between the sizes of the two pairs of lobes, indicating that an episode of jet realignment took place only very recently. Based on all the available radio data for 3C 293, we have performed a detailed spectral modelling for the older and younger lobes in the system. In this way we derived the lobes' ages and jet energetics, which we then compared to the accretion power in the source. We found that the 200 kpc-scale outer lobes of 3C 293 are ~60 Myr old and that jet activity related to the formation of the outer lobes ceased within the last Myr. Meanwhile, the inner 4 kpc-scale lobes, tilted by ~40 deg with respect to the outer ones, are only about ~0.3 ...

  19. The crystal structure of the secreted aspartic protease 1 from Candida parapsilosis in complex with pepstatin A

    Energy Technology Data Exchange (ETDEWEB)

    Dostál, Ji& #345; í; Brynda, Ji& #345; í; Hrušková-Heidingsfeldová, Olga; Sieglová, Irena; Pichová, Iva; & #344; ezá& #269; ová, Pavlína; (ASCR-ICP)

    2010-09-01

    Opportunistic pathogens of the genus Candida cause infections representing a major threat to long-term survival of immunocompromised patients. Virulence of the Candida pathogens is enhanced by production of extracellular proteolytic enzymes and secreted aspartic proteases (Saps) are therefore studied as potential virulence factors and possible targets for therapeutic drug design. Candida parapsilosis is less invasive than C. albicans, however, it is one of the leading causative agents of yeast infections. We report three-dimensional crystal structure of Sapp1p from C. parapsilosis in complex with pepstatin A, the classical inhibitor of aspartic proteases. The structure of Sapp1p was determined from protein isolated from its natural source and represents the first structure of Sap from C. parapsilosis. Overall fold and topology of Sapp1p is very similar to the archetypic fold of monomeric aspartic protease family and known structures of Sap isoenzymes from C. albicans and Sapt1p from C. tropicalis. Structural comparison revealed noticeable differences in the structure of loops surrounding the active site. This resulted in differential character, shape, and size of the substrate binding site explaining divergent substrate specificities and inhibitor affinities. Determination of structures of Sap isoenzymes from various species might contribute to the development of new Sap-specific inhibitors.

  20. Crystal structure of HIV-1 protease in situ product complex and observation of a low-barrier hydrogen bond between catalytic aspartates

    OpenAIRE

    Das, Amit; Prashar, Vishal; Mahale, Smita; Serre, L; Ferrer, J.-L.; Hosur, M. V.

    2006-01-01

    HIV-1 protease is an effective target for designing drugs against AIDS, and structural information about the true transition state and the correct mechanism can provide important inputs. We present here the three-dimensional structure of a bi-product complex between HIV-1 protease and the two cleavage product peptides AETF and YVDGAA. The structure, refined against synchrotron data to 1.65 Å resolution, shows the occurrence of the cleavage reaction in the crystal, with the product peptides st...

  1. Insights into Cleavage Specificity from the Crystal Structure of Foot-and-Mouth Disease Virus 3C Protease Complexed with a Peptide Substrate

    DEFF Research Database (Denmark)

    Zunszain, Patricia A; Knox, Stephen R; Sweeney, Trevor R;

    2010-01-01

    Foot-and-mouth disease (FMD) is a serious, widespread viral disease of cloven-hoofed animals, including important agricultural species such as cattle, sheep, pigs and goats (19, 45). The virus spreads rapidly and, although endemic and epidemic situations can be controlled using vaccines...... that are based on inactivated virus particles, political and technical difficulties with the maintenance and use of vaccine stocks has stimulated the search for alternative means of tackling the disease, such as anti-viral drugs (16). The development of such treatments will demand a detailed knowledge...

  2. Structural and functional characterization of complex formation between two Kunitz-type serine protease inhibitors from Russell's Viper venom.

    Science.gov (United States)

    Mukherjee, Ashis K; Dutta, Sumita; Kalita, Bhargab; Jha, Deepak K; Deb, Pritam; Mackessy, Stephen P

    2016-01-01

    Snake venom Kunitz-type serine protease inhibitors (KSPIs) exhibit various biological functions including anticoagulant activity. This study elucidates the occurrence and subunit stoichiometry of a putative complex formed between two KSPIs (Rusvikunin and Rusvikunin-II) purified from the native Rusvikunin complex of Pakistan Russell's Viper (Daboia russelii russelii) venom (RVV). The protein components of the Rusvikunin complex were identified by LC-MS/MS analysis. The non-covalent interaction between two major components of the complex (Rusvikunin and Rusvikunin-II) at 1:2 stoichiometric ratio to form a stable complex was demonstrated by biophysical techniques such as spectrofluorometric, classical gel-filtration, equilibrium gel-filtration, circular dichroism (CD), dynamic light scattering (DLS), RP-HPLC and SDS-PAGE analyses. CD measurement showed that interaction between Rusvikunin and Rusvikunin-II did not change their overall secondary structure; however, the protein complex exhibited enhanced hydrodynamic diameter and anticoagulant activity as compared to the individual components of the complex. This study may lay the foundation for understanding the basis of protein complexes in snake venoms and their role in pathophysiology of snakebite.

  3. Structural and biochemical characterization of the inhibitor complexes of xenotropic murine leukemia virus-related virus protease

    Energy Technology Data Exchange (ETDEWEB)

    Li, Mi; Gustchina, Alla; Matúz, Krisztina; Tözsér, Jozsef; Namwong, Sirilak; Goldfarb, Nathan E.; Dunn, Ben M.; Wlodawer, Alexander (Debrecen); (NCI); (Florida); (Suan Sunandha)

    2012-10-23

    Interactions between the protease (PR) encoded by the xenotropic murine leukemia virus-related virus and a number of potential inhibitors have been investigated by biochemical and structural techniques. It was observed that several inhibitors used clinically against HIV PR exhibit nanomolar or even subnanomolar values of K{sub i}, depending on the exact experimental conditions. Both TL-3, a universal inhibitor of retroviral PRs, and some inhibitors originally shown to inhibit plasmepsins were also quite potent, whereas inhibition by pepstatin A was considerably weaker. Crystal structures of the complexes of xenotropic murine leukemia virus-related virus PR with TL-3, amprenavir and pepstatin A were solved at high resolution and compared with the structures of complexes of these inhibitors with other retropepsins. Whereas TL-3 and amprenavir bound in a predictable manner, spanning the substrate-binding site of the enzyme, two molecules of pepstatin A bound simultaneously in an unprecedented manner, leaving the catalytic water molecule in place.

  4. Protein phosphatase complex PP5/PPP2R3C dephosphorylates P-glycoprotein/ABCB1 and down-regulates the expression and function.

    Science.gov (United States)

    Katayama, Kazuhiro; Yamaguchi, Miho; Noguchi, Kohji; Sugimoto, Yoshikazu

    2014-04-01

    P-glycoprotein (P-gp)/ABCB1 is a key molecule of multidrug resistance in cancer. Protein phosphatase (PP) 2A, regulatory subunit B, gamma (PPP2R3C), which is a regulatory subunit of PP2A and PP5, was identified as a binding candidate to P-gp. Immunoprecipitation-western blotting revealed that PP5 and PPP2R3C were coprecipitated with P-gp, while PP2A was not. PP5/PPP2R3C dephosphorylated protein kinase A/protein kinase C-phosphorylation of P-gp. Knockdown of PP5 and/or PPP2R3C increased P-gp expression and lowered the sensitivity to vincristine and doxorubicin. Consequently, our results indicate that PP5/PPP2R3C negatively regulates P-gp expression and function.

  5. Fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus aspartic protease, an Aspergillus aspartic protease per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a protease of the aspartic proteinase-type,

  6. MBL-associated serine protease-3 circulates in high serum concentrations predominantly in complex with Ficolin-3 and regulates Ficolin-3 mediated complement activation

    DEFF Research Database (Denmark)

    Skjoedt, Mikkel-Ole; Palarasah, Yaseelan; Munthe-Fog, Lea;

    2010-01-01

    The human lectin complement pathway (LCP) involves circulating complexes consisting of mannose-binding lectin (MBL) or ficolins in association with serine proteases named MASP-1, -2 and -3 and a non-enzymatic protein, sMAP. MASP-3 originates from the MASP1 gene through differential splicing and...

  7. Crystallisation and preliminary X-ray diffraction analysis of the protease from Southampton norovirus complexed with a Michael-acceptor inhibitor

    Energy Technology Data Exchange (ETDEWEB)

    Coates, Leighton [ORNL; Cooper, Jon [University of Southampton, England; Hussey, Robert [University of Southampton, England

    2008-01-01

    Noroviruses are the predominant cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional parts. Here, the crystallization of the recombinant protease from the Southampton norovirus is described. While the native crystals were found to diffract only to medium resolution (2.9 {angstrom}), cocrystals of an inhibitor complex diffracted X-rays to 1.7 {angstrom} resolution. The polypeptide inhibitor (Ac-EFQLQ-propenyl ethyl ester) possesses an amino-acid sequence designed to match the substrate specificity of the enzyme, but was synthesized with a reactive Michael acceptor group at the C-terminal end.

  8. High-resolution complex of papain with remnants of a cysteine protease inhibitor derived from Trypanosoma brucei

    OpenAIRE

    Alphey, Magnus S.; Hunter, William N.

    2006-01-01

    Attempts to cocrystallize the cysteine protease papain derived from the latex of Carica papaya with an inhibitor of cysteine proteases (ICP) from Trypanosoma brucei were unsuccessful. However, crystals of papain that diffracted to higher resolution, 1.5 Å, than other crystals of this archetypal cysteine protease were obtained, so the analysis was continued. Surprisingly, the substrate-binding cleft was occupied by two short peptide fragments which have been assigned as remnants of ICP. Compar...

  9. Crystal Structure of the VP4 Protease from Infectious Pancreatic Necrosis Virus Reveals the acyl-enzyme Complex for an Intermolecular Self-Cleavage Reaction

    Energy Technology Data Exchange (ETDEWEB)

    Lee,J.; Feldman, A.; Delmas, B.; Paetzel, M.

    2007-01-01

    Infectious pancreatic necrosis virus (IPNV), an aquatic birnavirus that infects salmonid fish, encodes a large polyprotein (NH{sub 2}-pVP2-VP4-VP3-COOH) that is processed through the proteolytic activity of its own protease, VP4, to release the proteins pVP2 and VP3. pVP2 is further processed to give rise to the capsid protein VP2 and three peptides that are incorporated into the virion. Reported here are two crystal structures of the IPNV VP4 protease solved from two different crystal symmetries. The electron density at the active site in the triclinic crystal form, refined to 2.2-{angstrom} resolution, reveals the acyl-enzyme complex formed with an internal VP4 cleavage site. The complex was generated using a truncated enzyme in which the general base lysine was substituted. Inside the complex, the nucleophilic Ser{sup 633}O{gamma} forms an ester bond with the main-chain carbonyl of the C-terminal residue, Ala{sup 716}, of a neighboring VP4. The structure of this substrate-VP4 complex allows us to identify the S1, S3, S5, and S6 substrate binding pockets as well as other substrate-VP4 interactions and therefore provides structural insights into the substrate specificity of this enzyme. The structure from the hexagonal crystal form, refined to 2.3-{angstrom} resolution, reveals the free-binding site of the protease. Three-dimensional alignment with the VP4 of blotched snakehead virus, another birnavirus, shows that the overall structure of VP4 is conserved despite a low level of sequence identity ({approx}19%). The structure determinations of IPNV VP4, the first of an acyl-enzyme complex for a Ser/Lys dyad protease, provide insights into the catalytic mechanism and substrate recognition of this type of protease.

  10. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

    Processing proteases are proteases which proteolytically activate proteins and peptides into their biologically active form. Processing proteases play an important role in biotechnology as tools in protein fusion technology. Fusion strategies where helper proteins or peptide tags are fused...... to the protein of interest are an elaborate method to optimize expression or purification systems. It is however critical that fusion proteins can be removed and processing proteases can facilitate this in a highly specific manner. The commonly used proteases all have substrate specificities to the N...... 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...

  11. Protease inhibitors as possible pitfalls in proteomic analyses of complex biological samples

    OpenAIRE

    Clifton, James; Huang, Feilei; Rucevic, Marijana; Cao, Lulu; Hixson, Douglas; Josic, Djuro

    2011-01-01

    Sample preparation, especially protein and peptide fractionation prior to identification by mass spectrometry (MS) are typically applied to reduce sample complexity. The second key element in this process is proteolytic digestion that is performed mostly by trypsin. Optimization of this step is an important factor in order to achieve both speed and better performance of proteomic analysis, and tryptic digestion prior to the MS analysis is topic of many studies. To date, only few studies pay a...

  12. Selective modulation of the CD4 molecular complex by Pseudomonas aeruginosa alkaline protease and elastase

    DEFF Research Database (Denmark)

    Pedersen, B K; Kharazmi, A; Theander, T G;

    1987-01-01

    The binding of monoclonal antibodies against CD4 was specifically inhibited by treatment of human CD4+ cells with either alkaline protease (AP) or elastase (Ela), purified from Pseudomonas aeruginosa. Binding of antibodies against CD3 (pan T), CD5 (pan T), CD8 (T suppressor/cytotoxic), HLA-ABC, HLA......-DR, HLA-DQ, HLA-DP/DR, and beta 2 microglobulin was not inhibited by AP or Ela. Heat-inactivation of the proteases at 65 degrees C for 20 min or treatment with the metal chelator EDTA abolished the inhibitory activity of both proteases. These findings may serve to develop novel immunological methods...

  13. Protease inhibitor

    DEFF Research Database (Denmark)

    2009-01-01

    The present invention relates to a polypeptide exhibiting a protease inhibitory activity and uses of said polypeptide in methods for inhibiting, directly or indirectly, one or more proteases of the blood clotting cascade. The invention also relates to use of said polypeptide as a pharmaceutical e...

  14. Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales.

    Science.gov (United States)

    Yokoyama, Hideshi; Matsui, Eriko; Hiramoto, Kana; Forterre, Patrick; Matsui, Ikuo

    2013-07-01

    The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein-protein complexes, which suggested the possibility of self-assembling of STOPP. Protein-protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin. PMID:23587725

  15. 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)

  16. Insights into the structural function of the complex of HIV-1 protease with TMC-126: molecular dynamics simulations and free-energy calculations

    Energy Technology Data Exchange (ETDEWEB)

    Li, Dan; Han, Ju-Guang; Chen, Hang; Li, Liang; Zhao, Run-Ning Zhao; Liu, Guang; Duan, Yuhua

    2012-05-01

    The binding properties of the protein-inhibitor complex of human immunodeficiency virus type 1 (HIV-1) protease with the inhibitor TMC-126 are investigated by combining computational alanine scanning (CAS) mutagenesis with binding free-energy decomposition (BFED). The calculated results demonstrate that the flap region (residues 38-58) and the active site region (residues 23-32) in HIV-1 protease contribute 63.72% of the protease to the binding of the inhibitor. In particular, the mechanisms for the interactions of key residues of these species are fully explored and analyzed. Interestingly, the regression analyses show that both CAS and BFED based on the generalized Born model yield similar results, with a correlation coefficient of 0.94. However, compared to CAS, BFED is faster and can decompose the per-residue binding free-energy contributions into backbone and sidechain contributions. The results obtained in this study are useful for studying the binding mechanism between receptor and ligand and for designing potent inhibitors that can combat diseases.

  17. Molecular modeling and molecular dynamics simulation study of the human Rab9 and RhoBTB3 C-terminus complex

    Science.gov (United States)

    Junaid, Muhammad; Muhseen, Ziyad Tariq; Ullah, Ata; Wadood, Abdul; Liu, Junjun; Zhang, Houjin

    2014-01-01

    Rab9 is required for the transport of mannose 6-phosphate receptors to the trans-Golgi network from late endosomes through the interaction with its effector: RhoBTB3. Earlier research indicates the C-terminus of RhoBTB3 (Rho_Cterm) is used for the interaction with Rab9. We used the homology modeling along with the molecular dynamics (MD) simulation to study the binding pattern of Rho_Cterm and Rab9 at atomic level. Both modeled structures, Rab9 and Rho_Cterm, are of high quality as suggested by the Ramachandran plot and ProCheck. The complex of Rab9-Rho_Cterm was generated by unrestrained pairwise docking using ZDOCK server. The interface of complex is consistent with the previous experimental data. The results of MD simulation indicate that the binding interface is stable along the simulation process. PMID:25670879

  18. Crystallization of a Nonclassical Kazal-type Carcinoscorpius Rotundicauda Serine Protease Inhibitor, CrSPI-1, Complexed with Subtilisin

    Energy Technology Data Exchange (ETDEWEB)

    Tulsidas, S.; Thangamani, S; Ho, B; Sivaraman, J; Ding, J

    2009-01-01

    Serine proteases play a major role in host-pathogen interactions. The innate immune system is known to respond to invading pathogens in a nonspecific manner. The serine protease cascade is an essential component of the innate immune system of the horseshoe crab. The serine protease inhibitor CrSPI isoform 1 (CrSPI-1), a unique nonclassical Kazal-type inhibitor of molecular weight 9.3 kDa, was identified from the hepatopancreas of the horseshoe crab Carcinoscorpius rotundicauda. It potently inhibits subtilisin and constitutes a powerful innate immune defence against invading microbes. Here, the cloning, expression, purification and cocrystallization of CrSPI-1 with subtilisin are reported. The crystals diffracted to 2.6 {angstrom}resolution and belonged to space group P2{sub 1}, with unit-cell parameters a = 73.8, b = 65.0, c = 111.9 {angstrom}, {beta} = 95.4. The Matthews coefficient (VM = 2.64 {angstrom}3 Da-1, corresponding to 53% solvent content) and analysis of the preliminary structure solution indicated the presence of one heterotrimer (1:2 ratio of CrSPI-1:subtilisin) and one free subtilisin molecule in the asymmetric unit.

  19. Developpement of a photoaffinity probe for the sensitive detection of matrix metallo-protease active forms from complex biological systems

    International Nuclear Information System (INIS)

    A new activity-based probe able to covalently modify the active site of proteases belonging to the matrix metallo-protease family (MMPs) has been developed in this thesis project. The probe was shown to behave as potent inhibitor of several MMPs, with nanomolar Ki values. This probe was also able to modify specifically only the free active site of MMPs, with particular high yields of cross-linking varying from 50 % to 11 %, depending of the MMPs tested. Using radioactivity as means of detection, this probe was able to detect active form of MMPs with a threshold of 1 femto-mole. Applied to the study of bronchoalveolar fluids (BAL) from mice exposed to nanoparticles by a lung aspiration protocol, this probe revealed the presence of the catalytic domain of MMP-12 under its active form, but not in control animals. When used to detect active form of MMPs from extracts obtained from human arteries of patient suffering from atherosclerosis, the probe was not able to detect such MMP active forms. Despite this negative result, the detection of active form of MMP in pathological fluid like BAL has never been reported before this work. Having validated this novel MMP activity-based probe, it will be possible to use it now for detecting MMPs from other pathological fluids or tissues extracts in which MMPs can be good markers of the pathology. (author)

  20. Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex

    International Nuclear Information System (INIS)

    The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

  1. Insights into the mechanism of drug resistance: X-ray structure analysis of G48V/C95F tethered HIV-1 protease dimer/saquinavir complex

    Energy Technology Data Exchange (ETDEWEB)

    Prashar, Vishal; Bihani, Subhash C.; Das, Amit [Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India); Rao, D.R. [Manufacturing and Research Division, CIPLA Ltd., L.B.S. Marg, Vikhroli, Mumbai 400083 (India); Hosur, M.V., E-mail: hosur@barc.gov.in [Solid State Physics Division, Bhabha Atomic Research Centre, Trombay, Mumbai 400085 (India)

    2010-06-11

    The mutation G48V in HIV-1 protease is a major resistance mutation against the drug saquinavir. Recently, G48V mutation is found to co-exist with the mutation C95F in AIDS patients treated with saquinavir. We report here the three-dimensional crystal structure of G48V/C95F tethered HIV-1 protease/saquinavir complex. The structure indicates following as the possible causes of drug resistance: (1) loss of direct van der Waals interactions between saquinavir and enzyme residues PHE-53 and PRO-1081, (2) loss of water-mediated hydrogen bonds between the carbonyl oxygen atoms in saquinavir and amide nitrogen atoms of flap residues 50 and 1050, (3) changes in inter-monomer interactions, which could affect the energetics of domain movements associated with inhibitor-binding, and (4) significant reduction in the stability of the mutant dimer. The present structure also provides a rationale for the clinical observation that the resistance mutations C95F/G48V/V82A occur as a cluster in AIDS patients.

  2. INTEGRATED POLARIZATION PROPERTIES OF 3C48, 3C138, 3C147, AND 3C286

    Energy Technology Data Exchange (ETDEWEB)

    Perley, R. A.; Butler, B. J., E-mail: RPerley@nrao.edu, E-mail: BButler@nrao.edu [National Radio Astronomy Observatory, P.O. Box O, Socorro, NM 87801 (United States)

    2013-06-01

    We present the integrated polarization properties of the four compact radio sources 3C48, 3C138, 3C147, and 3C286, from 1 to 50 GHz, over a 30 yr time frame spanning 1982-2012. Using the polarized emission of Mars, we have determined that the position angle of the linearly polarized emission of 3C286 rises from 33 Degree-Sign at 8 GHz to 36 Degree-Sign at 45 GHz. There is no evidence for a change in the position angle over time. Using these values, the position angles of the integrated polarized emission from the other three sources are determined as a function of frequency and time. The fractional polarization of 3C286 is found to be slowly rising, at all frequencies, at a rate of {approx}0.015% yr{sup -1}. The fractional polarizations of 3C48, 3C138, and 3C147 are all slowly variable, with the variations correlated with changes in the total flux densities of these sources.

  3. Bacterial IgA protease-mediated degradation of agIgA1 and agIgA1 immune complexes as a potential therapy for IgA Nephropathy.

    Science.gov (United States)

    Wang, Li; Li, Xueying; Shen, Hongchun; Mao, Nan; Wang, Honglian; Cui, Luke; Cheng, Yuan; Fan, Junming

    2016-01-01

    Mesangial deposition of aberrantly glycosylated IgA1 (agIgA1) and its immune complexes is a key pathogenic mechanism of IgA nephropathy (IgAN). However, treatment of IgAN remains ineffective. We report here that bacteria-derived IgA proteases are capable of degrading these pathogenic agIgA1 and derived immune complexes in vitro and in vivo. By screening 14 different bacterial strains (6 species), we found that 4 bacterial IgA proteases from H. influenzae, N. gonorrhoeae and N. meningitidis exhibited high cleaving activities on serum agIgA1 and artificial galactose-depleted IgA1 in vitro and the deposited agIgA1-containing immune complexes in the mesangium of renal biopsy from IgAN patients and in a passive mouse model of IgAN in vitro. In the modified mouse model of passive IgAN with abundant in situ mesangial deposition of the agIgA-IgG immune complexes, a single intravenous delivery of IgA protease from H. influenzae was able to effectively degrade the deposited agIgA-IgG immune complexes within the glomerulus, demonstrating a therapeutic potential for IgAN. In conclusion, the bacteria-derived IgA proteases are biologically active enzymes capable of cleaving the circulating agIgA and the deposited agIgA-IgG immune complexes within the kidney of IgAN. Thus, the use of such IgA proteases may represent a novel therapy for IgAN. PMID:27485391

  4. 3C DUCT DESIGN METHOD

    Institute of Scientific and Technical Information of China (English)

    Huan-RueiShiu; Feng-ChuOu; Sih-LiChen

    2002-01-01

    A new 3C duct design method is proposed for designing a high quality, energy-efficiency cost-effective air duct system. It not only considers the demand of volume flow rate, but also takes into consideration a number of issues including system pressure balance, noise, vibration, space limitation and total system cost. This new method comprises three major calculation procedures:initial computer-aided design (CAD), computer-aided simulation (CAS) and correction processes (CP). An example is presented in this study to understand the characteristics of 3C method. It shows that 3C duct design method provides a simple computation procedure for an optimum air duct system. It also shortens the design schedule, prevents human calculation errors, and reduces the dependence on designer experience. In addition to apply in a new duct system design, 3C duct design method is also a powerful design tool for the expansion of an existing duct system.

  5. Palmitoylation of protease-activated receptor-1 regulates adaptor protein complex-2 and -3 interaction with tyrosine-based motifs and endocytic sorting.

    Science.gov (United States)

    Canto, Isabel; Trejo, JoAnn

    2013-05-31

    Protease-activated receptor-1 (PAR1) is a G protein-coupled receptor for the coagulant protease thrombin. Thrombin binds to and cleaves the N terminus of PAR1, generating a new N terminus that functions as a tethered ligand that cannot diffuse away. In addition to rapid desensitization, PAR1 trafficking is critical for the regulation of cellular responses. PAR1 displays constitutive and agonist-induced internalization. Constitutive internalization of unactivated PAR1 is mediated by the clathrin adaptor protein complex-2 (AP-2), which binds to a distal tyrosine-based motif localized within the C-terminal tail (C-tail) domain. Once internalized, PAR1 is sorted from endosomes to lysosomes via AP-3 interaction with a second C-tail tyrosine motif proximal to the transmembrane domain. However, the regulatory processes that control adaptor protein recognition of PAR1 C-tail tyrosine-based motifs are not known. Here, we report that palmitoylation of PAR1 is critical for regulating proper utilization of tyrosine-based motifs and endocytic sorting. We show that PAR1 is basally palmitoylated at highly conserved C-tail cysteines. A palmitoylation-deficient PAR1 mutant is competent to signal and exhibits a marked increase in constitutive internalization and lysosomal degradation compared with wild type receptor. Intriguingly, enhanced constitutive internalization of PAR1 is mediated by AP-2 and requires the proximal tyrosine-based motif rather than the distal tyrosine motif used by wild type receptor. Moreover, palmitoylation-deficient PAR1 displays increased degradation that is mediated by AP-3. These findings suggest that palmitoylation of PAR1 regulates appropriate utilization of tyrosine-based motifs by adaptor proteins and endocytic trafficking, processes that are critical for maintaining appropriate expression of PAR1 at the cell surface. PMID:23580642

  6. Protease gene families in Populus and Arabidopsis

    Directory of Open Access Journals (Sweden)

    Jansson Stefan

    2006-12-01

    Full Text Available Abstract Background Proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. Similarities and differences between the proteases expressed in different species may give valuable insights into their physiological roles and evolution. Results We have performed a comparative analysis of protease genes in the two sequenced dicot genomes, Arabidopsis thaliana and Populus trichocarpa by using genes coding for proteases in the MEROPS database 1 for Arabidopsis to identify homologous sequences in Populus. A multigene-based phylogenetic analysis was performed. Most protease families were found to be larger in Populus than in Arabidopsis, reflecting recent genome duplication. Detailed studies on e.g. the DegP, Clp, FtsH, Lon, rhomboid and papain-Like protease families showed the pattern of gene family expansion and gene loss was complex. We finally show that different Populus tissues express unique suites of protease genes and that the mRNA levels of different classes of proteases change along a developmental gradient. Conclusion Recent gene family expansion and contractions have made the Arabidopsis and Populus complements of proteases different and this, together with expression patterns, gives indications about the roles of the individual gene products or groups of proteases.

  7. Insight to structural subsite recognition in plant thiol protease-inhibitor complexes : Understanding the basis of differential inhibition and the role of water

    Directory of Open Access Journals (Sweden)

    Mukhopadhayay Bishnu P

    2001-09-01

    Full Text Available Abstract Background This work represents an extensive MD simulation / water-dynamics studies on a series of complexes of inhibitors (leupeptin, E-64, E-64-C, ZPACK and plant cysteine proteases (actinidin, caricain, chymopapain, calotropin DI of papain family to understand the various interactions, water binding mode, factors influencing it and the structural basis of differential inhibition. Results The tertiary structure of the enzyme-inhibitor complexes were built by visual interactive modeling and energy minimization followed by dynamic simulation of 120 ps in water environment. DASA study with and without the inhibitor revealed the potential subsite residues involved in inhibition. Though the interaction involving main chain atoms are similar, critical inspection of the complexes reveal significant differences in the side chain interactions in S2-P2 and S3-P3 pairs due to sequence differences in the equivalent positions of respective subsites leading to differential inhibition. Conclusion The key finding of the study is a conserved site of a water molecule near oxyanion hole of the enzyme active site, which is found in all the modeled complexes and in most crystal structures of papain family either native or complexed. Conserved water molecules at the ligand binding sites of these homologous proteins suggest the structural importance of the water, which changes the conventional definition of chemical geometry of inhibitor binding domain, its shape and complimentarity. The water mediated recognition of inhibitor to enzyme subsites (Pn...H2O....Sn of leupeptin acetyl oxygen to caricain, chymopapain and calotropinDI is an additional information and offer valuable insight to potent inhibitor design.

  8. Complement component 3 (C3)

    Science.gov (United States)

    ... page: //medlineplus.gov/ency/article/003539.htm Complement component 3 (C3) To use the sharing features on this page, ... be some throbbing. Why the Test is Performed C3 and C4 are the most commonly measured complement components. A complement test may be used to monitor ...

  9. 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.

  10. Mouse Ficolin B Has an Ability to Form Complexes with Mannose-Binding Lectin-Associated Serine Proteases and Activate Complement through the Lectin Pathway

    Directory of Open Access Journals (Sweden)

    Yuichi Endo

    2012-01-01

    Full Text Available Ficolins are thought to be pathogen-associated-molecular-pattern-(PAMP- recognition molecules that function to support innate immunity. Like mannose-binding lectins (MBLs, most mammalian ficolins form complexes with MBL-associated serine proteases (MASPs, leading to complement activation via the lectin pathway. However, the ability of murine ficolin B, a homologue of human M-ficolin, to perform this function is still controversial. The results of the present study show that ficolin B in mouse bone marrow is an oligomeric protein. Ficolin B, pulled down using GlcNAc-agarose, contained very low, but detectable, amounts of MASP-2 and small MBL-associated protein (sMAP and showed detectable C4-deposition activity on immobilized N-acetylglucosamine. These biochemical features of ficolin B were confirmed using recombinant mouse ficolin B produced in CHO cells. Taken together, these results suggest that like other mammalian homologues, murine ficolin B has an ability to exert its function via the lectin pathway.

  11. 复合蛋白酶在卤鹅类潮式卤水生产中的应用%Application of Protease Complex in the Production of Chaozhou Brine Goose

    Institute of Scientific and Technical Information of China (English)

    陈宇; 郭卓钊; 郭美媛; 郭奕纯; 杨曼; 黄妙云

    2012-01-01

    针对蛋白酶在潮式卤鹅传统熟肉食品生产中的应用,以剪切力的变化来研究木瓜蛋白酶、胰蛋白酶和复合蛋白酶对卤鹅肉质的影响.通过单因素试验和正交试验确定了最佳的工艺生产条件为:复合酶液的胰蛋白酶用量为2000 U/kg原料、木瓜蛋白酶用量为1000 U/kg原料,温度为20℃,处理时间为30 min,所得到的卤鹅颜色酱红有光泽、肉质细嫩有弹性,滋味浓郁.%This paper described the application of protease complex in the traditional Chaozhou brine goose, by analysis of the change of shearing force after dealing by papain, trypsin and mixed proteases. The optional conditions were obtained by orthogonal experiments as follows: trypsin in mixed proteases 2000 U/kg, papain in mixed proteases 1000 U/kg, temperature 20 ℃and time 30 min. The brine goose had a glossy garnet red color, and the meat showed a delicious and elastic taste with rich flavor.

  12. Control of natural transformation in salivarius Streptococci through specific degradation of σX by the MecA-ClpCP protease complex.

    Science.gov (United States)

    Wahl, Astrid; Servais, Florence; Drucbert, Anne-Sophie; Foulon, Catherine; Fontaine, Laetitia; Hols, Pascal

    2014-08-01

    Competence for natural DNA transformation is a tightly controlled developmental process in streptococci. In mutans and salivarius species, the abundance of the central competence regulator σ(X) is regulated at two levels: transcriptional, by the ComRS signaling system via the σ(X)/ComX/SigX-inducing peptide (XIP), and posttranscriptional, by the adaptor protein MecA and its associated Clp ATPase, ClpC. In this study, we further investigated the mechanism and function of the MecA-ClpC control system in the salivarius species Streptococcus thermophilus. Using in vitro approaches, we showed that MecA specifically interacts with both σ(X) and ClpC, suggesting the formation of a ternary σ(X)-MecA-ClpC complex. Moreover, we demonstrated that MecA ultimately targets σ(X) for its degradation by the ClpCP protease in an ATP-dependent manner. We also identify a short sequence (18 amino acids) in the N-terminal domain of σ(X) as essential for the interaction with MecA and subsequent σ(X) degradation. Finally, increased transformability of a MecA-deficient strain in the presence of subinducing XIP concentrations suggests that the MecA-ClpCP proteolytic complex acts as an additional locking device to prevent competence under inappropriate conditions. A model of the interplay between ComRS and MecA-ClpCP in the control of σ(X) activity is proposed. PMID:24837292

  13. Investigations with Protease.

    Science.gov (United States)

    Yip, Din Yan

    1997-01-01

    Presents two simple and reliable ways for measuring protease activity that can be used for a variety of investigations in a range of biology class levels. The investigations use protease from a variety of sources. (DDR)

  14. 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...

  15. Regulation of protease-activated receptor 1 signaling by the adaptor protein complex 2 and R4 subfamily of regulator of G protein signaling proteins.

    Science.gov (United States)

    Chen, Buxin; Siderovski, David P; Neubig, Richard R; Lawson, Mark A; Trejo, Joann

    2014-01-17

    The G protein-coupled protease-activated receptor 1 (PAR1) is irreversibly proteolytically activated by thrombin. Hence, the precise regulation of PAR1 signaling is important for proper cellular responses. In addition to desensitization, internalization and lysosomal sorting of activated PAR1 are critical for the termination of signaling. Unlike most G protein-coupled receptors, PAR1 internalization is mediated by the clathrin adaptor protein complex 2 (AP-2) and epsin-1, rather than β-arrestins. However, the function of AP-2 and epsin-1 in the regulation of PAR1 signaling is not known. Here, we report that AP-2, and not epsin-1, regulates activated PAR1-stimulated phosphoinositide hydrolysis via two different mechanisms that involve, in part, a subset of R4 subfamily of "regulator of G protein signaling" (RGS) proteins. A significantly greater increase in activated PAR1 signaling was observed in cells depleted of AP-2 using siRNA or in cells expressing a PAR1 (420)AKKAA(424) mutant with defective AP-2 binding. This effect was attributed to AP-2 modulation of PAR1 surface expression and efficiency of G protein coupling. We further found that ectopic expression of R4 subfamily members RGS2, RGS3, RGS4, and RGS5 reduced activated PAR1 wild-type signaling, whereas signaling by the PAR1 AKKAA mutant was minimally affected. Intriguingly, siRNA-mediated depletion analysis revealed a function for RGS5 in the regulation of signaling by the PAR1 wild type but not the AKKAA mutant. Moreover, activation of the PAR1 wild type, and not the AKKAA mutant, induced Gαq association with RGS3 via an AP-2-dependent mechanism. Thus, AP-2 regulates activated PAR1 signaling by altering receptor surface expression and through recruitment of RGS proteins. PMID:24297163

  16. Protease-activated Receptor-4 Signaling and Trafficking Is Regulated by the Clathrin Adaptor Protein Complex-2 Independent of β-Arrestins.

    Science.gov (United States)

    Smith, Thomas H; Coronel, Luisa J; Li, Julia G; Dores, Michael R; Nieman, Marvin T; Trejo, JoAnn

    2016-08-26

    Protease-activated receptor-4 (PAR4) is a G protein-coupled receptor (GPCR) for thrombin and is proteolytically activated, similar to the prototypical PAR1. Due to the irreversible activation of PAR1, receptor trafficking is intimately linked to signal regulation. However, unlike PAR1, the mechanisms that control PAR4 trafficking are not known. Here, we sought to define the mechanisms that control PAR4 trafficking and signaling. In HeLa cells depleted of clathrin by siRNA, activated PAR4 failed to internalize. Consistent with clathrin-mediated endocytosis, expression of a dynamin dominant-negative K44A mutant also blocked activated PAR4 internalization. However, unlike most GPCRs, PAR4 internalization occurred independently of β-arrestins and the receptor's C-tail domain. Rather, we discovered a highly conserved tyrosine-based motif in the third intracellular loop of PAR4 and found that the clathrin adaptor protein complex-2 (AP-2) is important for internalization. Depletion of AP-2 inhibited PAR4 internalization induced by agonist. In addition, mutation of the critical residues of the tyrosine-based motif disrupted agonist-induced PAR4 internalization. Using Dami megakaryocytic cells, we confirmed that AP-2 is required for agonist-induced internalization of endogenous PAR4. Moreover, inhibition of activated PAR4 internalization enhanced ERK1/2 signaling, whereas Akt signaling was markedly diminished. These findings indicate that activated PAR4 internalization requires AP-2 and a tyrosine-based motif and occurs independent of β-arrestins, unlike most classical GPCRs. Moreover, these findings are the first to show that internalization of activated PAR4 is linked to proper ERK1/2 and Akt activation. PMID:27402844

  17. Theoretical study of the structure, bonding and electronic behaviour of sandwich complexes [M3(C7H7)2X3]- (M = Ni, Pd, Pt; X = F, Cl)

    Science.gov (United States)

    Zhou, Ke; Min, Suotian; Xue, Ganglin; Huang, Wendeng

    2014-08-01

    The correlations between the structural and electronic properties of the clusters [M3X3]3- and sandwich complexes [M3(C7H7)2X3]- (where M = Ni, Pd, Pt; X = F, Cl) were studied with density functional theory (B3PW91). All of the sandwich complexes are donating and back-donating metal-ligand bonding structures. The influence of the ligand, significant variations in the Msbnd C, Msbnd X, Msbnd M, Csbnd C bond lengths and binding energies were examined to obtain qualitative and quantitative pictures of the intramolecular C7R7+-M3X33- interactions. Our theoretical investigations show that the binding energies of the sandwich complexes gradually reduce from Ni to Pt, as well as from F to Cl. Meanwhile, the geometric and electronic structures and the relative stabilities have a strong relation to each other. S1). 7-B-(H-10) is a delocalised σ orbital, which has contributions from Pt (15% s orbital of each Pt, 8% d orbital of each Pt), and there is little contribution from Cl (Table S2). 7-C-(H-11) is a delocalised π orbital, which mainly involves contribution from Pt (25% dxz and dyz of each Pt) and Cl (8.5% pz of each Cl) (Table S3). 7-D-(H-14) is a delocalised σ orbital, which is mostly composed of d orbitals (14% for each Pt) and p orbitals (16% for each Cl) (Table S4). Pt s (10%) and d (10%) orbitals mainly participate in the formations of 7-E-(H-23), and there is a 13% p orbital contribution from each Cl (Table S5). For structure 13, the LUMO is mainly s orbitals of C, and there is some contribution from the s and d orbitals of Pt (Table S6), whereas the HOMO is mainly s orbitals of C (Table S7). 13-c-(H-14) is mainly the dxz and dyz orbitals of Pt (Table S8).NICS can be used to predict and understand some of the properties of a molecule, especially its stability due to aromatic stabilisation, which is based on the negative of the magnetic shielding computed at or above the geometrical centres of rings or clusters. Systems with negative NICS values are aromatic

  18. Poliovirus 2Apro induces the nucleic translocation of poliovirus 3CD and 3C' proteins

    Institute of Scientific and Technical Information of China (English)

    Wenwu Tian; Zongqiang Cui; Zhiping Zhang; Hongping Wei; XianEn Zhang

    2011-01-01

    Poliovirus genomic RNA replication, protein translation, and virion assembly are performed in the cytoplasm of host cells. However, this does not mean that there is no relationship between poliovirus infection and the cellular nucleus. In this study, recombinant fluorescence-tagged poliovirus 3CD and 3C' proteins were shown to be expressed mainly in the cytoplasm of Vero cells in the absence of other viral proteins. However, upon poliovirus infection, many of these proteins redistributed to the nucleus, as well as to the cytoplasm. A series of transfec-tion experiments revealed that the poliovirus 2Apro was responsible for the same redistribution of 3CD and 3C' proteins to the nucleus. Furthermore, a mutant 2Apr0 protein lacking protease activity abrogated this effect The poliovirus 2Apro protein was also found to co-localize with the IN up 153 protein, a component of the nuclear pore complexes on the nuclear envelope. These data provide further evidence that there are intrinsic interactions between poliovirus proteins and the cell nucleus, despite that many processes in the poliovirus replication cycle occur in the cytoplasm.

  19. Lung protease/anti-protease network and modulation of mucus production and surfactant activity.

    Science.gov (United States)

    Garcia-Verdugo, Ignacio; Descamps, Delphyne; Chignard, Michel; Touqui, Lhousseine; Sallenave, Jean-Michel

    2010-11-01

    Lung epithelium guarantees gas-exchange (performed in the alveoli) and protects from external insults (pathogens, pollutants…) present within inhaled air. Both functions are facilitated by secretions lining airway surface liquid, mucus (in the upper airways) and pulmonary surfactant (in the alveoli). Mucins, the main glycoproteins present within the mucus, are responsible for its rheologic properties and participate in lung defense mechanisms. In parallel, lung collectins are pattern recognition molecules present in pulmonary surfactant that also modulate lung defense. During chronic airways diseases, excessive protease activity can promote mucus hypersecretion and degradation of lung collectins and therefore contribute to the pathophysiology of these diseases. Importantly, secretion of local and systemic anti-proteases might be crucial to equilibrate the protease/anti-protease unbalance and therefore preserve the function of lung host defense compounds and airway surface liquid homeostasis. In this review we will present information relative to proteases able to modulate mucin production and lung collectin integrity, two important compounds of innate immune defense. One strategy to preserve physiological mucus production and collectin integrity during chronic airways diseases might be the over-expression of local 'alarm' anti-proteases such as SLPI and elafin. Interestingly, a cross-talk between lung collectins and anti-protease activity has recently been described, implicating the presence within the lung of a complex network between proteases, anti-proteases and pattern recognition molecules, which aims to keep or restore homeostasis in resting or inflamed lungs. PMID:20493919

  20. Degradation of phycobilisomes in Synechocystis sp. PCC6803: evidence for essential formation of an NblA1/NblA2 heterodimer and its codegradation by A Clp protease complex.

    Science.gov (United States)

    Baier, Antje; Winkler, Wiebke; Korte, Thomas; Lockau, Wolfgang; Karradt, Anne

    2014-04-25

    When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3-5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ~7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used "model organism" Synechocystis sp. PCC6803 expresses two such genes, nblA16803 and nblA26803, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N2-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.

  1. Novel fungal protease.

    NARCIS (Netherlands)

    Buxton, F.; Jarai, G.; Visser, J.

    1994-01-01

    The present invention concerns a novel DNA sequence coding for an Aspergillus serine protease of the subtilisin-type, an Aspergillus serine protease of the subtilisin-type per se and a method for the preparation thereof. The invention further concerns a novel Aspergillus mutant strain defective in a

  2. Native Elution of Yeast Protein Complexes Obtained by Affinity Capture.

    Science.gov (United States)

    LaCava, John; Fernandez-Martinez, Javier; Rout, Michael P

    2016-01-01

    This protocol describes two options for the native (nondenaturing) elution of protein complexes obtained by affinity capture. The first approach involves the elution of complexes purified through a tag that includes a human rhinovirus 3C protease (PreScission protease) cleavage site sequence between the protein of interest and the tag. Incubation with the protease cleaves immobilized complexes from the affinity medium. The second approach involves the release of protein A-tagged protein complexes using a competitive elution reagent called PEGylOx. The degree of purity of the native assemblies eluted is sample dependent and strongly influenced by the affinity capture. It should be noted that the efficiency of native elution is commonly lower than that of elution by a denaturing agent (e.g., SDS) and the release of the complex will be limited by the activity of the protease or the inhibition constant (Ki) of the competitive release agent. However, an advantage of native release is that some nonspecifically bound materials tend to stay adsorbed to the affinity medium, providing an eluted fraction of higher purity. Finally, keep in mind that the presence of the protease or elution peptide could potentially affect downstream applications; thus, their removal should be considered. PMID:27371597

  3. Enteroviral proteases: structure, host interactions and pathogenicity.

    Science.gov (United States)

    Laitinen, Olli H; Svedin, Emma; Kapell, Sebastian; Nurminen, Anssi; Hytönen, Vesa P; Flodström-Tullberg, Malin

    2016-07-01

    Enteroviruses are common human pathogens, and infections are particularly frequent in children. Severe infections can lead to a variety of diseases, including poliomyelitis, aseptic meningitis, myocarditis and neonatal sepsis. Enterovirus infections have also been implicated in asthmatic exacerbations and type 1 diabetes. The large disease spectrum of the closely related enteroviruses may be partially, but not fully, explained by differences in tissue tropism. The molecular mechanisms by which enteroviruses cause disease are poorly understood, but there is increasing evidence that the two enteroviral proteases, 2A(pro) and 3C(pro) , are important mediators of pathology. These proteases perform the post-translational proteolytic processing of the viral polyprotein, but they also cleave several host-cell proteins in order to promote the production of new virus particles, as well as to evade the cellular antiviral immune responses. Enterovirus-associated processing of cellular proteins may also contribute to pathology, as elegantly demonstrated by the 2A(pro) -mediated cleavage of dystrophin in cardiomyocytes contributing to Coxsackievirus-induced cardiomyopathy. It is likely that improved tools to identify targets for these proteases will reveal additional host protein substrates that can be linked to specific enterovirus-associated diseases. Here, we discuss the function of the enteroviral proteases in the virus replication cycle and review the current knowledge regarding how these proteases modulate the infected cell in order to favour virus replication, including ways to avoid detection by the immune system. We also highlight new possibilities for the identification of protease-specific cellular targets and thereby a way to discover novel mechanisms contributing to disease. Copyright © 2016 John Wiley & Sons, Ltd. PMID:27145174

  4. Comparative study of the catalytic activity of the complexes Cp{sup *}RuCl(PAr{sub 3}){sub 2} [Ar = -C{sub 6H}5 and 4-CF{sub 3}-C{sub 6}H{sub 4}] in the ATRP of styrene

    Energy Technology Data Exchange (ETDEWEB)

    Villa-Hernandez, Alejandro M.; Rosales-Velazquez, Claudia P.; Torres-Lubian, Jose R., E-mail: rtorres@ciqa.mx [Departamento de Sintesis de Polimeros, Centro de Investigacion en Quimica Aplicada, Coah. (Mexico); Saldivar-Guerra, Enrique [Departamento de Procesos de Polimerizacion, Centro de Investigacion en Quimica Aplicada, Coah. (Mexico)

    2011-09-15

    Styrene polymerization by ATRP was conducted independently using the complexes Cp{sup *}RuCl(PPh{sub 3}){sub 2}, and Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2} as catalysts, in order to evaluate the influence of the electronic properties of the phosphine ligands on the rate and control of the polymerization. The kinetic data for polymerizations carried out with Cp{sup *}RuCl(PPh{sub 3}){sub 2}, show that molecular weights increase linearly with conversion with an average initiation efficiency of 0.77. The molecular weights obtained in the kinetic study with Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2} also increase with conversion but show a marked deviation below the theoretical molecular weights. This behavior was explained by the gradual, irreversible, oxidation of catalyst Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2} as confirmed by {sup 31}P-NMR spectroscopy. Catalyst Cp{sup *}RuCl(PPh{sub 3}){sub 2} promotes the polymerization with a rate of polymerization higher than that obtained using Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2}; this is consistent with the better electron donating properties of PPh{sub 3} versus P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}. Preliminary studies of styrene polymerization by ATRP in supercritical CO{sub 2}, shows that only catalyst Cp{sup *}RuCl[P(4-CF{sub 3}-C{sub 6}H{sub 4}){sub 3}]{sub 2}, with fluorinated ligands, was active. (author)

  5. Structural Analysis of a Viral Ovarian Tumor Domain Protease from the Crimean-Congo Hemorrhagic Fever Virus in Complex with Covalently Bonded Ubiquitin

    Energy Technology Data Exchange (ETDEWEB)

    Capodagli, Glenn C.; McKercher, Marissa A.; Baker, Erica A.; Masters, Emily M.; Brunzelle, Joseph S.; Pegan, Scott D. (Denver); (NWU)

    2014-10-02

    Crimean-Congo hemorrhagic fever (CCHF) virus is a tick-borne, negative-sense, single-stranded RNA [ssRNA(-)] nairovirus that produces fever, prostration, and severe hemorrhages in humans. With fatality rates for CCHF ranging up to 70% based on several factors, CCHF is considered a dangerous emerging disease. Originally identified in the former Soviet Union and the Congo, CCHF has rapidly spread across large sections of Europe, Asia, and Africa. Recent reports have identified a viral homologue of the ovarian tumor protease superfamily (vOTU) within its L protein. This protease has subsequently been implicated in downregulation of the type I interferon immune response through cleavage of posttranslational modifying proteins ubiquitin (Ub) and the Ub-like interferon-simulated gene 15 (ISG15). Additionally, homologues of vOTU have been suggested to perform similar roles in the positive-sense, single-stranded RNA [ssRNA(+)] arteriviruses. By utilizing X-ray crystallographic techniques, the structure of vOTU covalently bound to ubiquitin propylamine, a suicide substrate of the enzyme, was elucidated to 1.7 {angstrom}, revealing unique structural elements that define this new subclass of the OTU superfamily. In addition, kinetic studies were carried out with aminomethylcoumarin (AMC) conjugates of monomeric Ub, ISG15, and NEDD8 (neural precursor cell expressed, developmentally downregulated 8) substrates in order to provide quantitative insights into vOTU's preference for Ub and Ub-like substrates.

  6. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

    Bacterial pathogens rely on proteolysis for variety of purposes during the infection process. In the cytosol, the main proteolytic players are the conserved Clp and Lon proteases that directly contribute to virulence through the timely degradation of virulence regulators and indirectly by providing...... 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...... 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...

  7. Tomato ringspot nepovirus protease: characterization and cleavage site specificity

    NARCIS (Netherlands)

    Hans, F.; Sanfacon, H.

    1995-01-01

    We have cloned the region of tomato ringspot nepovirus (TomRSV) RNA-1 coding for the putative TomRSV 3C-related protease (amino acids 1213 to 1508) in a transcription vector and in a transient expression vector. Using cell-free transcription and translation systems and plant protoplasts, we have dem

  8. 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.

  9. Subtilisin-like proteases in nematodes.

    Science.gov (United States)

    Poole, Catherine B; Jin, Jingmin; McReynolds, Larry A

    2007-09-01

    Cleavage by subtilisin-like proteases (subtilases) is an essential step in post-translational processing of proteins found in organisms ranging from yeast to mammals. Our knowledge of the diversity of this protease family in nematodes is aided by the rapid increase in sequence information, especially from the Brugia malayi genome project. Genetic studies of the subtilases in Caenorhabitis elegans give valuable insight into the biological function of these proteases in other nematode species. In this review, we focus on the subtilases in filarial nematodes as well as other parasitic and free-living nematodes in comparison to what is known in C. elegans. Topics to be addressed include expansion and diversity of the subtilase gene family during evolution, enhanced complexity created by alternative RNA splicing, molecular and biochemical characterization of the different subtilases and the challenges of designing subtilase-specific inhibitors for parasitic nematodes. PMID:17570539

  10. Well-defined mono(η3-allyl)nickel complex MONi(η3-C3H5) (M = Si or Al) grafted onto silica or alumina: A molecularly dispersed nickel precursor for syntheses of supported small size nickel nanoparticles

    KAUST Repository

    Li, Lidong

    2014-01-01

    Preparing evenly-dispersed small size nickel nanoparticles over inert oxides remains a challenge today. In this context, a versatile method to prepare supported small size nickel nanoparticles (ca. 1-3 nm) with narrow size distribution via a surface organometallic chemistry (SOMC) route is described. The grafted mono(η3-allyl)nickel complexes MONi(η 3-C3H5) (M = Si or Al) as precursors are synthesized and fully characterized by elemental analysis, FTIR spectroscopy and paramagnetic solid-state NMR. © 2014 the Partner Organisations.

  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. Nucleic Acid Aptamers Against Proteases

    OpenAIRE

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø; Andreasen, P A

    2011-01-01

    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...

  13. Proteolysis in Plastids of Arabidopsis Thaliana: Functional Analysis of ClpS1,2,T and their Physical and Genetic Interactions with the ClpPR Protease Core Complex and Clp Chaperones

    Energy Technology Data Exchange (ETDEWEB)

    van Wijk, Klaas

    2009-01-12

    Chloroplasts are essential organelles required for plant growth and biomass production. They synthesize many essential secondary metabolites (e.g. hormones, isoprenoids, amino acids, etc.) and house the photosynthetic apparatus needed for conversion of light energy and CO2 into chemical energy [in the form of reduced carbohydrates, ATP and NADPH]. Thus chloroplasts are essential for life on earth and essential for production of bioenergy. Formation and maintenance of a functional chloroplast requires an extensive investment in the biogenesis and homeostasis apparatus. Protease and proteolysis play a critical role in these processes, with the Clp gene family being particularly central. Proteolysis of proteins and protein complexes in plastids is poorly understood, and is not only critical for biogenesis, adaptation and maintenance but is also important for plant development. Several years ago, the vanWijk lab identified a large and relatively abundant ClpP/R/S complex, along with ClpC1,C2 and ClpD chaperones and a putative Clp affinity modulator in plastids. So far, no substrate recognition mechanism has been determined for any Clp complex in plants. The purpose of this grant was to initiate functional analysis of three members of the Clp family.

  14. Structure-Function of Falcipains: Malarial Cysteine Proteases

    Directory of Open Access Journals (Sweden)

    Kailash C. Pandey

    2012-01-01

    Full Text Available Evidence indicates that cysteine proteases play essential role in malaria parasites; therefore an obvious area of investigation is the inhibition of these enzymes to treat malaria. Studies with cysteine protease inhibitors and manipulating cysteine proteases genes have suggested a role for cysteine proteases in hemoglobin hydrolysis. The best characterized Plasmodium cysteine proteases are falcipains, which are papain family enzymes. Falcipain-2 and falcipain-3 are major hemoglobinases of P. falciparum. Structural and functional analysis of falcipains showed that they have unique domains including a refolding domain and a hemoglobin binding domain. Overall, the complexes of falcipain-2 and falcipain-3 with small and macromolecular inhibitors provide structural insight to facilitate the design or modification of effective drug treatment against malaria. Drug development targeting falcipains should be aided by a strong foundation of biochemical and structural studies.

  15. Synthesis of novel p-tert-butylcalix[4]arene Schiff bases and their complexes with C60, potential HIV-Protease inhibitors

    Science.gov (United States)

    Khadra, Khalid Abu; Mizyed, Shehadeh; Marji, Deeb; Haddad, Salim F.; Ashram, Muhammad; Foudeh, Ayat

    2015-02-01

    Some p-tert-butylcalix[4]arene Schiff base crown ethers were synthesized, characterized using 1H, 13C-NMR, DEPT 135 and Mass spectrometry. Their complexes with C60 were isolated and characterized. The inhibition effect of these complexes on HIVP was studied and found that complexes of 9 and 10 have comparable Ki values to Pepstatine which is known as HIVP inhibitor and used as a control. The synthesis of the ligands, complexes and the inhibition behavior are discussed in this article.

  16. Death proteases come alive

    NARCIS (Netherlands)

    Woltering, E.J.

    2004-01-01

    Cell death in plants exhibits morphological features comparable to caspase-mediated apoptosis in animals, suggesting that plant cell death is executed by (caspase-like) proteases. However, to date, no caspase homologues have been identified in plants and therefore the existence and nature of these p

  17. Proteases in Periodontal Disease

    Directory of Open Access Journals (Sweden)

    Ana Rita Sokolonski ANTON

    2006-09-01

    Full Text Available Introduction: The caries and the periodontal disease (PD are the most frequent alterations in the oral cavity. The PD presents two stages: gengivitis and periodontitis. The destruction of collagenous fibers which encases the tooth onto the alveolar bone is characteristic of the pariodontitis. The inclusion loss caused by this pathology is due to the presence of bacteria and their products, besides the tissue destruction. This process is caused by excessive discharge of cells of the organism defence which reach the damaged area, and among these cells are neutrophils. These cells free lysosomal granule, where enzymes known as proteases (elastase, colagenasis and catepsin G are present. When excessively delivered, they cause extensive tissue destruction. The organism innate defence respond to this process activating anti-proteases, such as alfa-1-antitripsin e alfa-2-macrogoblulin, and, as consequence, the inflammatory process is subdued. Objective: Revision of the literature on periodontitis and its markers. In periodontitis, the balance between protease and anti-protese seems to be altered and lead to the appearance of these ones. There is an increase of prevalence of PD in the world population. In recent times, it has been associated to systemic conditions that lead to tissue destruction. Perhaps, the cause is based on an exacerbated tissue reaction, more than on the bacterial aggression. Conclusion: The predisposition of the organism is an important factor for the disease development. At reading different studies, it was observed that the discharged protease during the neutrophils degranulation process has internal, not bacterial, origin.

  18. NATO-3C/Delta launch

    Science.gov (United States)

    1978-01-01

    NATO-3C, the third in a series of NATO defense-related communication satellites, is scheduled to be launched on a delta vehicle from the Eastern Test Range no earlier than November 15, 1978. NATO-3A and -3B were successfully launched by Delta vehicles in April 1976 and January 1977, respectively. The NATO-3C spacecraft will be capable of transmitting voice, data, facsimile, and telex messages among military ground stations. The launch vehicle for the NATO-3C mission will be the Delta 2914 configuration. The launch vehicle is to place the spacecraft in a synchronous transfer orbit. The spacecraft Apogee Kick motor is to be fired at fifth transfer orbit apogee to circularize its orbit at geosynchronous altitude of 35,900 km(22,260 miles) above the equator over the Atlantic Ocean somewhere between 45 and 50 degrees W longitude.

  19. The spectrum of 3C 58

    International Nuclear Information System (INIS)

    Digital spectra of three faint knots in the SNR 3C 58 have been obtained in the wavelength range lambdalambda4700--7300 with the intensified Reticon spectrometer at the 1.3 m telescope of McGraw-Hill Observatory. Emission lines of [S II], [N II], Hα, and [O III] were detected with radial velocities less than 100 km s-1. Although 3C 58 resembles the Crab Nebula in its radio properties and is thought to be the remnant of the supernova observed in A.D. 1181, the relative line intensities and radial velocities reported here more nearly resemble those of the Cygnus Loop and Kepler's SNR

  20. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

    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......-specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  1. Sumo-dependent substrate targeting of the SUMO protease Ulp1

    Directory of Open Access Journals (Sweden)

    Westerbeck Jason W

    2011-10-01

    Full Text Available Abstract Background In the yeast Saccharomyces cerevisiae, the essential small ubiquitin-like modifier (SUMO protease Ulp1 is responsible for both removing SUMO/Smt3 from specific target proteins and for processing precursor SUMO into its conjugation-competent form. Ulp1 localizes predominantly to nuclear pore complexes but has also been shown to deconjugate sumoylated septins at the bud-neck of dividing cells. How Ulp1 is directed to bud-neck localized septins and other cytoplasmic deconjugation targets is not well understood. Results Using a structure/function approach, we set out to elucidate features of Ulp1 that are required for substrate targeting. To aid our studies, we took advantage of a catalytically inactive mutant of Ulp1 that is greatly enriched at the septin ring of dividing yeast cells. We found that the localization of Ulp1 to the septins requires both SUMO and specific structural features of Ulp1's catalytic domain. Our analysis identified a 218-amino acid, substrate-trapping mutant of the catalytic domain of Ulp1, Ulp1(3(C580S, that is necessary and sufficient for septin localization. We also used the targeting and SUMO-binding properties of Ulp1(3(C580S to purify Smt3-modified proteins from cell extracts. Conclusions Our study provides novel insights into how the Ulp1 SUMO protease is actively targeted to its substrates in vivo and in vitro. Furthermore, we found that a substrate-trapping Ulp1(3(C580S interacts robustly with human SUMO1, SUMO2 and SUMO2 chains, making it a potentially useful tool for the analysis and purification of SUMO-modified proteins.

  2. Novel 2-oxoimidazolidine-4-carboxylic acid derivatives as Hepatitis C virus NS3-4A serine protease inhibitors: synthesis, activity, and X-ray crystal structure of an enzyme inhibitor complex

    Energy Technology Data Exchange (ETDEWEB)

    Arasappan, Ashok; Njoroge, F. George; Parekh, Tejal N.; Yang, Xiaozheng; Pichardo, John; Butkiewicz, Nancy; Prongay, Andrew; Yao, Nanhua; Girijavallabhan, Viyyoor (SPRI)

    2008-06-30

    Synthesis and HCV NS3 serine protease inhibitory activity of some novel 2-oxoimidazolidine-4-carboxylic acid derivatives are reported. Inhibitors derived from this new P2 core exhibited activity in the low {micro}M range. X-ray structure of an inhibitor, 15c bound to the protease is presented.

  3. L3+C air shower array

    CERN Multimedia

    Laurent Guiraud

    2000-01-01

    Photo 01: a view of the L3+C air shower array; 50 scintillators on the roof of the SX-hall above L3. Photo 02: view of one of the detectors of the array.Photo 04: detectors seen against the background of the LEP Point 2 facilities.

  4. PROTEASES AND PROTEASE INHIBITORS INTERACTION: DEFENCE STRATEGY AGAINST

    Directory of Open Access Journals (Sweden)

    R.S.DHANDE 1 N.J.CHIKHALE 2

    2014-12-01

    Full Text Available ABSTRACT: An increase in crop yield, its management and preservation are among the main challenges standing before the human population that exceed 10 billion by the mid of 21 st  century.  Every year, considerable agricultural losses occur due to repeated practices of cultivation of large genetically similar populations.  Such cultivation practices favors incidence of more insect pests (Hilder and Boulter, 1999;  Oerke  et  al.,  1994;  Smith,  1999.  To  solve  these  problems,  current approaches  rely  on  use  of  synthetic  chemicals  like  fertilizers,  insecticides, herbicides,  fungicides  etc.  But  this  exerts  excessively  high  pressure  on environment  and  destabilizes  the  ecological  balance.  The  traditional  pest control method involves the use of conventional pesticides, most of which are non-specific and wipe out the entire community, pollutes the agro-ecosystem, and  increases  the  cost  of  production.  The  emergence  of  gene  transfer technology  has  solved  some  problems  regarding  overuse  of  chemical pesticides.  The  delta  endotoxin  encoding  gene  from  Bacillus  thuringiensis,  a gram positive soil borne bacteria transferred in crops has given little relief from coleopterans and lepidopterans attack.  Whereas, the insects belonging to these orders like Helicoverpa Sps. have developed resistance against Bt toxins. The other approach takes advantage of use of plant genes encoding defense proteins like protease inhibitors which is more appealing, simpler and safer (Dunaevsky et.  al.,  2005.  Proteinase  inhibitors  (PIs  are  naturally  occurring  proteins  in living organisms and are able to inhibit & control the activity of proteases. PIs act  on  an  active  site  of  digestive  proteolytic  enzymes  and  form  a  stable complex  unlike  enzyme-substrate  or  enzyme-product  weak  complexes  which

  5. From proteases to proteomics.

    Science.gov (United States)

    Neurath, H

    2001-04-01

    This personal and professional autobiography covers the 50-yr period of 1950-2000 and includes the following topics: History of the University of Washington School of Medicine and its Department of Biochemistry (Mount Rainier and the University of Washington, recruiting faculty, biology, research programs); scientific editing (publication, Biochemistry, Protein Science, electronic publication); Europe revisited (Heidelberg, approaching retirement, the German Research Center, reunion in Vienna); and 50 yr of research on proteolytic enzymes (trypsin, carboxypeptidases, mast cell proteases, future developments).

  6. Substrate modulation of enzyme activity in the herpesvirus protease family

    OpenAIRE

    Lazic, Ana; Goetz, David H.; Nomura, Anson M.; Marnett, Alan B.; Craik, Charles S.

    2007-01-01

    The herpesvirus proteases are an example in which allosteric regulation of an enzyme activity is achieved through the formation of quaternary structure. Here, we report a 1.7 Å resolution structure of Kaposi’s Sarcoma herpesvirus protease in complex with a hexapeptide transition state analogue that stabilizes the dimeric state of the enzyme. Extended substrate binding sites are induced upon peptide binding. In particular, 104 Å2 of surface are buried in the newly formed S4 pocket when tyrosin...

  7. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior

    DEFF Research Database (Denmark)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu;

    2016-01-01

    -ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its CDR-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to...

  8. Observations with a VLB array. III. The sources 3C 120, 3C 273B, 2134+004, and 3C 84

    International Nuclear Information System (INIS)

    The sources 3C 120, 3C 273B, 2134+004, and 3C 84 have been observed at several epochs at 2.8 cm using a multielement very-long-baseline interferometer (VLBI). The resulting visibility data show that the brightness distributions of each source are variable. There are large changes in the visibilities of 3C 120 and 3C 273B on a time scale of months. For 3C 120, and perhaps 3C 273B, the changes can be explained by source distributions in which the components are separating at high (probably relativistic) speeds

  9. Protease-mediated drug delivery

    Science.gov (United States)

    Dickson, Eva F.; Goyan, Rebecca L.; Kennedy, James C.; Mackay, M.; Mendes, M. A. K.; Pottier, Roy H.

    2003-12-01

    Drugs used in disease treatment can cause damage to both malignant and normal tissue. This toxicity limits the maximum therapeutic dose. Drug targeting is of high interest to increase the therapeutic efficacy of the drug without increasing systemic toxicity. Certain tissue abnormalities, disease processes, cancers, and infections are characterized by high levels of activity of specific extracellular and/or intracellular proteases. Abnormally high activity levels of specific proteases are present at sites of physical or chemical trauma, blood clots, malignant tumors, rheumatoid arthritis, inflammatory bowel disease, gingival disease, glomerulonerphritis, and acute pancreatitis. Abnormal protease activity is suspected in development of liver thrombosis, pulmonary emphysema, atherosclerosis, and muscular dystrophy. Inactiviating disease-associated proteases by the administration of appropriate protease inhibitors has had limited success. Instead, one could use such proteases to target drugs to treat the condition. Protease mediated drug delivery offers such a possibility. Solubilizing groups are attached to insoluble drugs via a polypeptide chain which is specifically cleavable by certian proteases. When the solubilized drug enounters the protease, the solubilizing moieties are cleaved, and the drug precipitates at the disease location. Thus, a smaller systemic dosage could result in a therapeutic drug concentration at the treatment site with less systemic toxicity.

  10. Toxoplasma gondii aspartic protease 1 is not essential in tachyzoites.

    Science.gov (United States)

    Polonais, Valerie; Shea, Michael; Soldati-Favre, Dominique

    2011-08-01

    Aspartic proteases are important virulence factors for pathogens and are recognized as attractive drug targets. Seven aspartic proteases (ASPs) have been identified in Toxoplasma gondii genome. Bioinformatics and phylogenetic analyses regroup them into five monophyletic groups. Among them, TgASP1, a coccidian specific aspartic protease related to the food vacuole plasmepsins, is associated with the secretory pathway in non-dividing cells and relocalizes in close proximity to the nascent inner membrane complex (IMC) of daughter cells during replication. Despite a potential role for TgASP1 in IMC formation, the generation of a conventional knockout of the TgASP1 gene revealed that this protease is not required for T. gondii tachyzoite survival or for proper IMC biogenesis.

  11. Metal-based antimicrobial protease inhibitors.

    Science.gov (United States)

    Kellett, A; Prisecaru, A; Slator, C; Molphy, Z; McCann, M

    2013-01-01

    Limitations associated with the production cost, metabolic instability, side-effects, resistance and poor pharmacokinetics of organic protease inhibitors (PIs), which form an essential component of the front line HAART treatment for HIV, have fuelled efforts into finding novel, transition metal-based alternatives. Some of the attractive features of metalbased therapeutics include synthetic simplicity, solubility control, redox capability, expansion of coordination number and topography matching of the complex to the protein's active site. Building asymmetry into the complex, which may offer better discrimination between host and rogue cell, can readily be achieved through coordination of chiral ligands to the metal centre. Although the scope of this review has been limited to metal-based agents that have been reported to bind/inhibit HIV-1 and parasitic proteases, some desirables, such as high activity, low dosage, minimal toxicity, crossinhibition, unique binding modes and selectivity, have already been delivered. The variability of the d-block metals, coupled with the availability of designer organic ligands, augers well for the future development of clinical metallo-drugs for deployment against protease-associated, fatal diseases.

  12. Autoprocessing: an essential step for expression and purification of enterovirus 71 3C(pro) in Escherichia coli.

    Science.gov (United States)

    Huang, Shuqiong; Lyu, Yanning; Qing, Xianyun; Wang, Weiwei; Tang, Liang; Cheng, Kedi; Wang, Wei

    2013-11-01

    A gene encoding the 3BC of human enterovirus 71 (EV71) was cloned and inserted into a derivative of plasmid pET-32a(+) driven by T7 promoter. The expressed 3C protease (3C(pro)) autocatalytically cleaved itself from the recombinant protein Trx-3BC and the mature 3C(pro) partitioned in the soluble fraction of bacterial lysate. The 13-amino-acid peptide substrates with the junction of 3B/3C were used to verify the proteolysis activity of the purified 3C(pro). The EV71 3C(pro) had a Km value of 63 μM (measured by a continuous fluorescence assay). The other solid-phase activity assay of the EV71 3C(pro) was developed using HPLC to analyze the proteolytic products. The combination of two activity assays contributes to promote the identification of the specific inhibitors targeted to the EV71 3C(pro). PMID:23881322

  13. 汞和金属离子及多硫化物对木瓜蛋白酶活性的抑制作用%Inhibition of cysteine protease papain by metal ions and polysulfide complexes, especially mercuric ion

    Institute of Scientific and Technical Information of China (English)

    姜军; 杨晓达; 王夔

    2007-01-01

    Abstract: Aim Cysteine proteases are closely associated with many human and non-human pathological processes and are potential targets for metal ions especially Hg2+ and the related species. In the present work, on the basis of to the general study on the effects of some metal ions on the activity of papain, a well-known representative of cysteine protease family, the inhibitory effects of Hg2+ and polysulfide complexes were studied. Results All the metal ions tested (Hg2+, Cu2+, Ag+, Au3+, Zn2+, Cd2+, Fe3+, Mn2+, Pb2+, Yb3+) inhibit the activity of papain anda good correlation between the inhibitory potency and softness-and-hardness was observed. Among the metals, Hg2+ was shown to be a potent inhibitor of papain with a Ki of 2 × 10-7 mol· L-1 among. Excessive amounts of glutathione and cysteine could reactivate the enzyme activity of papain deactivated by Hg2+. These evidences supported that Hg2+ might bind to the catalytic site of papain. Interestingly,Hg (Ⅱ) polysulfide complexes were for the first time found to inhibit papain with a Ki of 7 × 10-6 mol ·L-1, whose potency is close to a well known mercury compound, thimerosal (Ki=2.7 × 10-6). In addition, Hg (Ⅱ) polysulfide complexes exhibit good permeability (1.9 × 10-5cm·s-1) to caco-2 monolayer. Conclusion These results suggested that mercury polysulfide complexes might be potential bioactive species in the interaction with cysteine proteases and other- SH-content proteins, providing a new clue to understand the mechanism of the toxicological and pharmacological actions of cinnabar and other insoluble mercury compounds.%目的 半胱氨酸蛋白酶参与了很多动植物生理过程和病理过程,是Hg2+及其化合物作用的潜在靶点.木瓜蛋白酶是半胱氨酸蛋白酶家族中最具代表性、研究最为广泛深入的一种蛋白酶,本文以木瓜蛋白酶为模型,研究了汞等金属离子及其化合物对半胱氨酸蛋白酶活性的抑制作用.结果 Hg2+对木瓜蛋白酶

  14. Double Faraday rotation toward 3C 27

    Science.gov (United States)

    Goldstein, S. J., Jr.; Reed, J. A.

    1984-08-01

    From observations of the integrated flux of 3C 27 with the NRAO 140 foot (43 m) telescope at 40 frequencies between 1250 and 1445 MHz, the authors deduce rotation measures of 165±15 and -104±4 rad m-2. Since the source (assumed to be a radio galaxy) has components 45arcsec apart, it is concluded that the net magnetic field reverses between these directions. One explanation is that a large magnetic field surrounding the central galaxy of the distant source covers one component but not the other. Another explanation is that our Galaxy contains a dipole field with a scale of order 1 pc. One component of the distant source is seen inside the current loop associated with the dipole field, while the other is seen outside the loop.

  15. 3C236 Radio Source, Interrupted?

    CERN Document Server

    O'Dea, C P; Baum, S A; Sparks, W B; Martel, A R; Allen, M G; Macchetto, F D; Miley, G K; Dea, Christopher P. O'; Koekemoer, Anton M.; Baum, Stefi A.; Sparks, William B.; Martel, Andre R.; Allen, Mark G.; Macchetto, Ferdinando D.; Miley, George K.

    2001-01-01

    We present new HST STIS/MAMA near-UV images and archival WFPC2 V and R band images which reveal the presence of four star forming regions in an arc along the edge of the dust lane in the giant (4 Mpc) radio galaxy 3C236. Two of the star forming regions are relatively young with ages of order 1E7 yr, while the other two are older with ages of order 1E8 - 1E9 yr which is comparable to the estimated age of the giant radio source. Based on dynamical and spectral aging arguments, we suggest that the fuel supply to the AGN was interrupted for 1E7 yr and has now been restored, resulting in the formation of the inner 2 kpc scale radio source. This time scale is similar to that of the age of the youngest of the star forming regions. We suggest that the transport of gas in the disk is non-steady and that this produces both the multiple episodes of star formation in the disk as well as the multiple epochs of radio source activity. If the inner radio source and the youngest star forming region are related by the same eve...

  16. Photometric reverberation mapping of 3C120

    CERN Document Server

    Nuñez, F Pozo; Westhues, C; Bruckmann, C; Haas, M; Chini, R; Steenbrugge, K; Murphy, M; 10.1051/0004-6361/201219107

    2013-01-01

    We present the results of a five month monitoring campaign of the local active galactic nuclei (AGN) 3C120. Observations with a median sampling of two days were conducted with the robotic 15cm telescope VYSOS-6 located near Cerro Armazones in Chile. Broad band (B,V) and narrow band (NB) filters were used in order to measure fluxes of the AGN and the H_beta broad line region (BLR) emission line. The NB flux is constituted by about 50% continuum and 50% H_beta emission line. To disentangle line and continuum flux, a synthetic H_beta light curve was created by subtracting a scaled V-band light curve from the NB light curve. Here we show that the H_beta emission line responds to continuum variations with a rest frame lag of 23.6 +/- 1.69 days. We estimate a virial mass of the central black hole M_BH = 57 +/- 27 * 10^6 solar masses, by combining the obtained lag with the velocity dispersion of a single contemporaneous spectrum. Using the flux variation gradient (FVG) method, we determined the host galaxy subtracte...

  17. Protease-Sensitive Synthetic Prions

    OpenAIRE

    Colby, David W; Rachel Wain; Baskakov, Ilia V.; Giuseppe Legname; Palmer, Christina G.; Nguyen, Hoang-Oanh B.; Azucena Lemus; Cohen, Fred E.; Stephen J DeArmond; Prusiner, Stanley B.

    2010-01-01

    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, ...

  18. Hydrothermal synthesis and study of an inorganic-organic hybrid vanadate of a nickel(II) coordination complex with pyrazine, Ni{sub 3}(C{sub 4}H{sub 4}N{sub 2}){sub 3}(V{sub 8}O{sub 23})

    Energy Technology Data Exchange (ETDEWEB)

    Larrea, Edurne S. [Dpto. de Mineralogia y Petrologia, Facultad de Ciencia y Tecnologia, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, UPV/EHU, Apdo. 644, E-48080 Bilbao (Spain); Mesa, Jose L., E-mail: joseluis.mesa@ehu.es [Dpto. de Quimica Inorganica, Facultad de Ciencia y Tecnologia, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, UPV/EHU, Apdo. 644, E-48080 Bilbao (Spain); Arriortua, Maria I. [Dpto. de Mineralogia y Petrologia, Facultad de Ciencia y Tecnologia, Universidad del Pais Vasco/Euskal Herriko Unibertsitatea, UPV/EHU, Apdo. 644, E-48080 Bilbao (Spain)

    2011-06-15

    Highlights: {yields} A novel inorganic-organic hybrid vanadate of nickel(II) coordination complex with pyrazine has been synthesized hydrothermally. {yields} The thermal and spectroscopic behavior has been studied. {yields} The compound shows AFM interactions which has been fitted to a magnetic model of lineal chains. -- Abstract: The three-dimensional hybrid compound Ni{sub 3}(C{sub 4}H{sub 4}N{sub 2}){sub 3}(V{sub 8}O{sub 23}) has been synthesized by mild hydrothermal methods under autogenous pressure at 170 {sup o}C. The structure of the phase is stable until 380 {sup o}C. The removal of the pyrazine molecules from the structure induces its collapse. The IR spectrum shows the vibration modes of the pyrazine molecule and those of the [VO{sub 4}]{sup 3-} groups. A UV-visible spectrum shows the characteristic bands of the Ni(II) d{sup 8}-high-spin cation in a slightly distorted octahedral coordination. Magnetic measurements indicate the existence of antiferromagnetic interactions that can be fitted with a chain model to give g = 2.31, J/k = -5.3, and zJ'/k = -5.5.

  19. Photosynthesis of C3, C3-C4, and C4 grasses at glacial CO2.

    Science.gov (United States)

    Pinto, Harshini; Sharwood, Robert E; Tissue, David T; Ghannoum, Oula

    2014-07-01

    Most physiology comparisons of C3 and C4 plants are made under current or elevated concentrations of atmospheric CO2 which do not reflect the low CO2 environment under which C4 photosynthesis has evolved. Accordingly, photosynthetic nitrogen (PNUE) and water (PWUE) use efficiency, and the activity of the photosynthetic carboxylases [Rubisco and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] were compared in eight C4 grasses with NAD-ME, PCK, and NADP-ME subtypes, one C3 grass, and one C3-C4 grass grown under ambient (400 μl l(-1)) and glacial (180 μl l(-1)) CO2. Glacial CO2 caused a smaller reduction of photosynthesis and a greater increase of stomatal conductance in C4 relative to C3 and C3-C4 species. Panicum bisulcatum (C3) acclimated to glacial [CO2] by doubling Rubisco activity, while Rubisco was unchanged in Panicum milioides (C3-C4), possibly due to its high leaf N and Rubisco contents. Glacial CO2 up-regulated Rubisco and PEPC activities in concert for several C4 grasses, while NADP-ME and PEP-CK activities were unchanged, reflecting the high control exerted by the carboxylases relative to the decarboxylases on the efficiency of C4 metabolism. Despite having larger stomatal conductance at glacial CO2, C4 species maintained greater PWUE and PNUE relative to C3-C4 and C3 species due to higher photosynthetic rates. Relative to other C4 subtypes, NAD-ME and PEP-CK grasses had the highest PWUE and PNUE, respectively; relative to C3, the C3-C4 grass had higher PWUE and similar PNUE at glacial CO2. Biomass accumulation was reduced by glacial CO2 in the C3 grass relative to the C3-C4 grass, while biomass was less reduced in NAD-ME grasses compared with NADP-ME and PCK grasses. Under glacial CO2, high resource use efficiency offers a key evolutionary advantage for the transition from C3 to C4 photosynthesis in water- and nutrient-limited environments.

  20. Serine proteases of parasitic helminths.

    Science.gov (United States)

    Yang, Yong; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-02-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we described the serine proteases that have been identified in parasitic helminths, including nematodes (Trichinella spiralis, T. pseudospiralis, Trichuris muris, Anisakis simplex, Ascaris suum, Onchocerca volvulus, O. lienalis, Brugia malayi, Ancylostoma caninum, and Steinernema carpocapsae), cestodes (Spirometra mansoni, Echinococcus granulosus, and Schistocephalus solidus), and trematodes (Fasciola hepatica, F. gigantica, and Schistosoma mansoni). Moreover, the possible biological functions of these serine proteases in the endogenous biological phenomena of these parasites and in the host-parasite interaction were also discussed. PMID:25748703

  1. Serine Proteases of Parasitic Helminths

    OpenAIRE

    Yong YANG; Wen, Yun jun; Cai, Ya Nan; Vallée, Isabelle; Boireau, Pascal; Liu, Ming Yuan; Cheng, Shi Peng

    2015-01-01

    Serine proteases form one of the most important families of enzymes and perform significant functions in a broad range of biological processes, such as intra- and extracellular protein metabolism, digestion, blood coagulation, regulation of development, and fertilization. A number of serine proteases have been identified in parasitic helminths that have putative roles in parasite development and nutrition, host tissues and cell invasion, anticoagulation, and immune evasion. In this review, we...

  2. The Resolved Outflow from 3C 48

    Science.gov (United States)

    Shih, Hsin-Yi; Stockton, Alan

    2014-10-01

    We investigate the properties of the high-velocity outflow driven by the young radio jet of 3C 48, a compact-steep-spectrum source. We use the Space Telescope Imaging Spectrograph on board the Hubble Space Telecope to obtain (1) low-resolution UV and optical spectra and (2) multi-slit medium-resolution spectra of the ionized outflow. With supporting data from ground-based spectrographs, we are able to accurately measure the ratios of diagnostic emission lines such as [O III] λ5007, [O III] λ3727, [N II] λ6548, Hα, Hβ, [Ne V] λ3425, and [Ne III] λ3869. We fit the observed emission-line ratios using a range of ionization models, powered by active galactic nucleus (AGN) radiation and shocks, produced by the MAPPINGS code. We have determined that AGN radiation is likely the dominant ionization source. The outflow's density is estimated to be in the range n = 103-104 cm-3, the mass is ~6 × 106 M ⊙, and the metallicity is likely equal to or higher than solar. Compared with the typical outflows associated with more evolved radio jets, this young outflow is denser, less massive, and more metal rich. Multi-slit observations allow us to construct a two-dimensional velocity map of the outflow that shows a wide range of velocities with distinct velocity components, suggesting a wide-angle clumpy outflow. Based in part on observations made with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the Association of Universities for Research in Astronomy, Inc., under NASA contract NAS 5-26555. These observations are associated with program GO-11574. Some of the data presented herein were obtained at the W. M. Keck Observatory, which is operated as a scientific partnership among the California Institute of Technology, the University of California and the National Aeronautics and Space Administration. The Observatory was made possible by the generous financial support of the W. M. Keck Foundation. Some of the

  3. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses

    Science.gov (United States)

    Yu, Jong W.; Hoffman, Sandy; Beal, Allison M.; Dykon, Angela; Ringenberg, Michael A.; Hughes, Anna C.; Dare, Lauren; Anderson, Amber D.; Finger, Joshua; Kasparcova, Viera; Rickard, David; Berger, Scott B.; Ramanjulu, Joshi; Emery, John G.; Gough, Peter J.; Bertin, John; Foley, Kevin P.

    2015-01-01

    CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo. PMID:25965667

  4. MALT1 Protease Activity Is Required for Innate and Adaptive Immune Responses.

    Directory of Open Access Journals (Sweden)

    Jong W Yu

    Full Text Available CARMA-BCL10-MALT1 signalosomes play important roles in antigen receptor signaling and other pathways. Previous studies have suggested that as part of this complex, MALT1 functions as both a scaffolding protein to activate NF-κB through recruitment of ubiquitin ligases, and as a protease to cleave and inactivate downstream inhibitory signaling proteins. However, our understanding of the relative importance of these two distinct MALT1 activities has been hampered by a lack of selective MALT1 protease inhibitors with suitable pharmacologic properties. To fully investigate the role of MALT1 protease activity, we generated mice homozygous for a protease-dead mutation in MALT1. We found that some, but not all, MALT1 functions in immune cells were dependent upon its protease activity. Protease-dead mice had defects in the generation of splenic marginal zone and peritoneal B1 B cells. CD4+ and CD8+ T cells displayed decreased T cell receptor-stimulated proliferation and IL-2 production while B cell receptor-stimulated proliferation was partially dependent on protease activity. In dendritic cells, stimulation of cytokine production through the Dectin-1, Dectin-2, and Mincle C-type lectin receptors was also found to be partially dependent upon protease activity. In vivo, protease-dead mice had reduced basal immunoglobulin levels, and showed defective responses to immunization with T-dependent and T-independent antigens. Surprisingly, despite these decreased responses, MALT1 protease-dead mice, but not MALT1 null mice, developed mixed inflammatory cell infiltrates in multiple organs, suggesting MALT1 protease activity plays a role in immune homeostasis. These findings highlight the importance of MALT1 protease activity in multiple immune cell types, and in integrating immune responses in vivo.

  5. CLIP proteases and Plasmodium melanization in Anopheles gambiae.

    Science.gov (United States)

    Barillas-Mury, Carolina

    2007-07-01

    Melanization is a potent immune response mediated by phenoloxidase (PO). Multiple Clip-domain serine proteases (CLIP) regulate PO activation as part of a complex cascade of proteases that are cleaved sequentially. The role of several CLIP as key activators or suppressors of the melanization responses of Anopheles gambiae to Plasmodium berghei (murine malaria) has been established recently using a genome-wide reverse genetics approach. Important differences in regulation of PO activation between An. gambiae strains were also identified. This review summarizes these findings and discusses our current understanding of the An. gambiae melanization responses to Plasmodium. PMID:17512801

  6. Microbial inhibitors of cysteine proteases.

    Science.gov (United States)

    Kędzior, Mateusz; Seredyński, Rafał; Gutowicz, Jan

    2016-08-01

    Cysteine proteases are one of the major classes of proteolytic enzymes involved in a number of physiological and pathological processes in plants, animals and microorganisms. When their synthesis, activity and localization in mammalian cells are altered, they may contribute to the development of many diseases, including rheumatoid arthritis, osteoporosis and cancer. Therefore, cysteine proteases have become promising drug targets for the medical treatment of these disorders. Inhibitors of cysteine proteases are also produced by almost every group of living organisms, being responsible for the control of intracellular proteolytic activity. Microorganisms synthesize cysteine protease inhibitors not only to regulate the activity of endogenous, often virulent enzymes, but also to hinder the host's proteolytic defense system and evade its immune responses against infections. Present work describes known to date microbial inhibitors of cysteine proteases in terms of their structure, enzyme binding mechanism, specificity and pathophysiological roles. The overview of both proteinaceous and small-molecule inhibitors produced by all groups of microorganisms (bacteria, archaea, fungi, protists) and viruses is provided. Subsequently, possible applications of microbial inhibitors in science, medicine and biotechnology are also highlighted. PMID:27048482

  7. Alpha-1 Antitrypsin Deficiency: Beyond the Protease/Antiprotease Paradigm.

    Science.gov (United States)

    Cosio, Manuel G; Bazzan, Erica; Rigobello, Chiara; Tinè, Mariaenrica; Turato, Graziella; Baraldo, Simonetta; Saetta, Marina

    2016-08-01

    From the discovery that alpha-1 antitrypsin (AAT) was an effective inhibitor of neutrophil elastase originated the classic paradigm of protease/antiprotease imbalance, linking lung destruction to the unopposed effect of proteases in patients with the deficiency. Notwithstanding its importance as an antiprotease, it has become evident that alpha-1 antitrypsin has important antiinflammatory and immune-regulatory activities, which may be critically involved in lung destruction. We review here recent evidence showing that, indeed, an important adaptive immune reaction is present in lungs with AAT deficiency, similar to the one seen in severe chronic obstructive pulmonary disease with normal AAT. On the basis of recent evidence from epidemiological, clinical, and pathogenetic studies, it is likely time to move on from the original protease/antiprotease hypothesis for the production of emphysema toward a more complex paradigm, involving the antiinflammatory and immune modulating functions of AAT. PMID:27564665

  8. Theoretical studies of the proton transfer behaviors in molecular complexes analogous to catalytic triad of serine protease: Toward understanding the existence and significance of the low-barrier hydrogen-bond in enzymatic catalysis

    Institute of Scientific and Technical Information of China (English)

    LI Ping; WANG WeiHua; BI SiWei; SONG Rui; BU YuXiang

    2009-01-01

    A representative acetate-(5-methylimidazole)-methanol system has been employed as a model of cata-lytic triad in serine protease to validate the formation processes of low-barrier H-bonds (LBHB) at the B3LYP/6-311++G** level of theory, and variable H-bonding characters from conventional ones to LBHBs have been represented along with the proceedings of proton transfer. Solvent effect is an important factor in modulation of the existence of an LBHB, where an LBHB (or a conventional H-bond) in the gas phase can be changed into a non-LBHB (an LBHB) upon solvation. The origin of the additional stabili-zation energy arising from the LBHB may be attributed to the H-bonding energy difference before and after proton transfer because the shared proton can freely move between the proton donor and proton acceptor. Most importantly, the order of magnitude of the stabilization energy depends on the studied systems. Furthermore, the nonexistence of LBHBs in the catalytic triad of serine proteases has been verified in a more sophisticated model treated using the ONIOM method. As a result, only the single proton transfer mechanism in the catalytic triad has been confirmed and the origin of the powerful catalytic efficiency of serine proteases should be attributed to other factors rather than the LBHB.

  9. Theoretical studies of the proton transfer behaviors in molecular complexes analogous to catalytic triad of serine protease:Toward understanding the existence and significance of the low-barrier hydrogen-bond in enzymatic catalysis

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    A representative acetate-(5-methylimidazole)-methanol system has been employed as a model of catalytic triad in serine protease to validate the formation processes of lowbarrier H-bonds(LBHB) at the B3LYP/6-311++G level of theory,and variable H-bonding characters from conventional ones to LBHBs have been represented along with the proceedings of proton transfer.Solvent effect is an important factor in modulation of the existence of an LBHB,where an LBHB(or a conventional H-bond) in the gas phase can be changed into a non-LBHB(an LBHB) upon solvation.The origin of the additional stabili-zation energy arising from the LBHB may be attributed to the H-bonding energy difference before and after proton transfer because the shared proton can freely move between the proton donor and proton acceptor.Most importantly,the order of magnitude of the stabilization energy depends on the studied systems.Furthermore,the nonexistence of LBHBs in the catalytic triad of serine proteases has been verified in a more sophisticated model treated using the ONIOM method.As a result,only the single proton transfer mechanism in the catalytic triad has been confirmed and the origin of the powerful catalytic efficiency of serine proteases should be attributed to other factors rather than the LBHB.

  10. Enzymatic Degradation of Ovalbumin by Various Proteases

    OpenAIRE

    Matsumoto, Kiyoshi; Yoshimaru, Tetsuro; Matsui, Toshiro; Osajima, Yutaka

    1997-01-01

    An investigation was made of the enzymatic hydrolysis of ovalbumin (OVA), a major allergen in egg white, by various acid and alkaline proteases. Protease YP-SS (acid protease) from Aspergillus niger and alcalase (alkaline protease) from BacilLus licheniformis were found to be useful for the degradation of OVA, respectively. OVA was almost totally hydrolyzed within 15 hr at 37℃ by alcalase. Alcalase acted rapidly to hydrolyze OVA, with about 90% of OVA being hydrolyzed within 30min., the react...

  11. 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.

  12. Exogenous proteases for meat tenderization.

    Science.gov (United States)

    Bekhit, Alaa A; Hopkins, David L; Geesink, Geert; Bekhit, Adnan A; Franks, Philip

    2014-01-01

    The use of exogenous proteases to improve meat tenderness has attracted much interest recently, with a view to consistent production of tender meat and added value to lower grade meat cuts. This review discusses the sources, characteristics, and use of exogenous proteases in meat tenderization to highlight the specificity of the proteases toward meat proteins and their impact on meat quality. Plant enzymes (such as papain, bromelain, and ficin) have been extensively investigated as meat tenderizers. New plant proteases (actinidin and zingibain) and microbial enzyme preparations have been of recent interest due to controlled meat tenderization and other advantages. Successful use of these enzymes in fresh meat requires their enzymatic kinetics and characteristics to be determined, together with an understanding of the impact of the surrounding environmental conditions of the meat (pH, temperature) on enzyme function. This enables the optimal conditions for tenderizing fresh meat to be established, and the elimination or reduction of any negative impacts on other quality attributes. PMID:24499119

  13. Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease

    Energy Technology Data Exchange (ETDEWEB)

    Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L.; Schiffer, Celia A. (CIT); (UMASS, MED)

    2012-10-23

    HIV-1 protease recognizes and cleaves more than 12 different substrates leading to viral maturation. While these substrates share no conserved motif, they are specifically selected for and cleaved by protease during viral life cycle. Drug resistant mutations evolve within the protease that compromise inhibitor binding but allow the continued recognition of all these substrates. While the substrate envelope defines a general shape for substrate recognition, successfully predicting the determinants of substrate binding specificity would provide additional insights into the mechanism of altered molecular recognition in resistant proteases. We designed a variant of HIV protease with altered specificity using positive computational design methods and validated the design using X-ray crystallography and enzyme biochemistry. The engineered variant, Pr3 (A28S/D30F/G48R), was designed to preferentially bind to one out of three of HIV protease's natural substrates; RT-RH over p2-NC and CA-p2. In kinetic assays, RT-RH binding specificity for Pr3 increased threefold compared to the wild-type (WT), which was further confirmed by isothermal titration calorimetry. Crystal structures of WT protease and the designed variant in complex with RT-RH, CA-p2, and p2-NC were determined. Structural analysis of the designed complexes revealed that one of the engineered substitutions (G48R) potentially stabilized heterogeneous flap conformations, thereby facilitating alternate modes of substrate binding. Our results demonstrate that while substrate specificity could be engineered in HIV protease, the structural pliability of protease restricted the propagation of interactions as predicted. These results offer new insights into the plasticity and structural determinants of substrate binding specificity of the HIV-1 protease.

  14. Full quantum mechanical study of binding of HIV-1 protease drugs

    Science.gov (United States)

    Zhang, Da W.; Zhang, John Z. H.

    Fully quantum mechanical studies of detailed binding interactions between HIV-1 protease and six FDA (Food and Drug Administration)-approved drugs (saquinavir, indinavir, ritonavir, nelfinavir, amprenavir, and lopinavir) are carried out using a recently developed MFCC (molecular fractionation with conjugate caps) method. The MFCC calculation produces a quantum mechanical interaction spectrum for any protease drug binding complex. Detailed quantitative analysis on binding of lopinavir to specific residues of the protease is given from the current study. The present calculation shows that the dominant binding of lopinavir to the protease is through the formation of a strong hydrogen bond between the central hydroxyl group of the drug to the aspartate oxygen of Asp25 in one of the two chains of the protease (A chain). This is closely followed by hydrogen binding of the drug to Asp29 in the B chain and somewhat weak hydrogen bonding to Asp30, Gly27, Gly48, and Ile50 in both chains. By partitioning all six drugs into four building blocks besides the central component containing the hydroxyl group, MFCC calculation finds that block III has essentially no binding interaction with the protease and the major binding interactions of these drugs are from blocks II and IV, in addition to the dominant central hydroxyl group. This detailed quantitative information on drug binding to the protease is very useful in rational design of new and improved inhibitors of HIV-1 protease and its mutants.

  15. Identification, synthesis and evaluation of SARS-CoV and MERS-CoV 3C-like protease inhibitors.

    Science.gov (United States)

    Kumar, Vathan; Tan, Kian-Pin; Wang, Ying-Ming; Lin, Sheng-Wei; Liang, Po-Huang

    2016-07-01

    Severe acute respiratory syndrome (SARS) led to a life-threatening form of atypical pneumonia in late 2002. Following that, Middle East Respiratory Syndrome (MERS-CoV) has recently emerged, killing about 36% of patients infected globally, mainly in Saudi Arabia and South Korea. Based on a scaffold we reported for inhibiting neuraminidase (NA), we synthesized the analogues and identified compounds with low micromolar inhibitory activity against 3CL(pro) of SARS-CoV and MERS-CoV. Docking studies show that a carboxylate present at either R(1) or R(4) destabilizes the oxyanion hole in the 3CL(pro). Interestingly, 3f, 3g and 3m could inhibit both NA and 3CL(pro) and serve as a starting point to develop broad-spectrum antiviral agents. PMID:27240464

  16. Genetic evidence supporting the association of protease and protease inhibitor genes with inflammatory bowel disease: a systematic review.

    Directory of Open Access Journals (Sweden)

    Isabelle Cleynen

    Full Text Available As part of the European research consortium IBDase, we addressed the role of proteases and protease inhibitors (P/PIs in inflammatory bowel disease (IBD, characterized by chronic mucosal inflammation of the gastrointestinal tract, which affects 2.2 million people in Europe and 1.4 million people in North America. We systematically reviewed all published genetic studies on populations of European ancestry (67 studies on Crohn's disease [CD] and 37 studies on ulcerative colitis [UC] to identify critical genomic regions associated with IBD. We developed a computer algorithm to map the 807 P/PI genes with exact genomic locations listed in the MEROPS database of peptidases onto these critical regions and to rank P/PI genes according to the accumulated evidence for their association with CD and UC. 82 P/PI genes (75 coding for proteases and 7 coding for protease inhibitors were retained for CD based on the accumulated evidence. The cylindromatosis/turban tumor syndrome gene (CYLD on chromosome 16 ranked highest, followed by acylaminoacyl-peptidase (APEH, dystroglycan (DAG1, macrophage-stimulating protein (MST1 and ubiquitin-specific peptidase 4 (USP4, all located on chromosome 3. For UC, 18 P/PI genes were retained (14 proteases and 4 protease inhibitors, with a considerably lower amount of accumulated evidence. The ranking of P/PI genes as established in this systematic review is currently used to guide validation studies of candidate P/PI genes, and their functional characterization in interdisciplinary mechanistic studies in vitro and in vivo as part of IBDase. The approach used here overcomes some of the problems encountered when subjectively selecting genes for further evaluation and could be applied to any complex disease and gene family.

  17. ATP-dependent protease in maize mitochondria

    International Nuclear Information System (INIS)

    ATP-dependent protease was identified in the matrix of Zea mays L. Sachara mitochondria. 14C-methylated casein has been used as a substrate, and the matrix ATP-dependent protease exhibited similar sensitivity towards specific inhibitors as the Lon protease from E. coli nd analogues from rat liver and yeast mitochondria. Here we report the existence of Lon like ATP-dependent protease in intact mitochondria prepared from 4-days-old epicotyls of Zea mays L. seedling. Enzyme has been purified from Lubrol treated mitochondria using ion exchange chromatography and gel filtration. The enzyme activity has been estimated using 14C-methylated casein as a substrate and sensitivity of the protease towards the specific inhibitors has been tested. ATP-dependent protease from the mitochondrial matrix of maize exhibit similar sensitivity to the above mentioned inhibitors like Lon protease from yeast and rat liver mitochondria as well as from E. coli. (authors)

  18. Biotechnology of Cold-Active Proteases

    Directory of Open Access Journals (Sweden)

    Tulasi Satyanarayana

    2013-05-01

    Full Text Available The bulk of Earth’s biosphere is cold (<5 °C and inhabited by psychrophiles. Biocatalysts from psychrophilic organisms (psychrozymes have attracted attention because of their application in the ongoing efforts to decrease energy consumption. Proteinases as a class represent the largest category of industrial enzymes. There has been an emphasis on employing cold-active proteases in detergents because this allows laundry operations at ambient temperatures. Proteases have been used in environmental bioremediation, food industry and molecular biology. In view of the present limited understanding and availability of cold-active proteases with diverse characteristics, it is essential to explore Earth’s surface more in search of an ideal cold-active protease. The understanding of molecular and mechanistic details of these proteases will open up new avenues to tailor proteases with the desired properties. A detailed account of the developments in the production and applications of cold-active proteases is presented in this review.

  19. Chaotic Feature in the Light Curve of 3C 273

    Institute of Scientific and Technical Information of China (English)

    Lei Liu

    2006-01-01

    Some nonlinear dynamical techniques, including state-space reconstruction and correlation integral, are used to analyze the light curve of 3C 273. The result is compared with a chaotic model. The similarities between them suggest there is a low-dimension chaotic attractor in the light curve of 3C 273.

  20. Role and efforts of T3C in corrosion economics

    Energy Technology Data Exchange (ETDEWEB)

    Perrigo, L.D.; Appleman, B.R.; Pamer, R.I.; Thompson, J.L.

    1979-11-01

    The basic purpose of T3C activity is to show how to acquire specific corrosion cost information so that overall costs for doing business can be reduced. The scope of T3C is to accumulate data, appraise methods, develop recommended practices, promote knowledge and communicate relative to the economic evaluation of corrosion and counter corrosion techniques.

  1. Role and efforts of T3C in corrosion economics

    International Nuclear Information System (INIS)

    The basic purpose of T3C activity is to show how to acquire specific corrosion cost information so that overall costs for doing business can be reduced. The scope of T3C is to accumulate data, appraise methods, develop recommended practices, promote knowledge and communicate relative to the economic evaluation of corrosion and counter corrosion techniques

  2. VLBI, MERLIN and HST observations of the giant radio galaxy 3C 236

    NARCIS (Netherlands)

    Schilizzi, RT; Tian, WW; Conway, JE; Nan, R; Miley, GK; Barthel, PD; Normandeau, M; Dallacasa, D; Gurvits, LI

    2001-01-01

    We present VLBI and MERLIN data at 1.66 and 4.99 GHz on the central component coincident with the nucleus of the giant radio galaxy, 3C 236. The nuclear radio structure is composed of two complexes of emission which are resolved on scales from 1 milli-arcsec (mas) to 1 arcsec. Oscillations with an a

  3. Reaction mechanism of -acylhydroxamate with cysteine proteases

    Indian Academy of Sciences (India)

    R Shankar; P Kolandaivel

    2007-09-01

    The gas-phase reaction mechanism of -acylhydroxamate with cysteine proteases has been investigated using ab initio and density functional theory. On the irreversible process, after breakdown of tetrahedral intermediate (INT1), small 1-2 anionotropic has been formed and rearranged to give stable by-products sulfenamide (P1) and thiocarbamate (P2) with considerable energy loss. While, on the reversible part of this reaction mechanism, intermediate (INT2) breaks down on oxidation, to form a stable product (P3). Topological and AIM analyses have been performed for hydrogen bonded complex in this reaction profile. Intrinsic reaction coordinates [IRC, minimum-energy path (MEP)] calculation connects the transition state between R-INT1, INT1-P1 and INT1-P2. The products P1, P2 and P3 are energetically more stable than the reactant and hence the reaction enthalpy is found to be exothermic.

  4. LPS counter regulates RNA expression of extracellular proteases and their inhibitors in murine macrophages

    DEFF Research Database (Denmark)

    Hald, Andreas; Rønø, Birgitte; Lund, Leif R;

    2012-01-01

    to extracellular matrix degradation. This process is accomplished by multiple proteolytic enzymes, including serine proteases and members of the matrix metalloproteinase family. In this study, we have utilized qPCR arrays to simultaneously analyze the temporal expression pattern of a range of genes involved...... was found for the genes encoding serine proteases, which were downregulated while their inhibitors were upregulated. In addition, intergenic comparison of the expression levels of related proteases revealed large differences in their basal expression level. These data highlight the complexity of the gene...

  5. Generation of a C3c specific monoclonal antibody and assessment of C3c as a putative inflammatory marker derived from complement factor C3

    DEFF Research Database (Denmark)

    Palarasah, Yaseelan; Skjødt, Karsten; Brandt, Jette;

    2010-01-01

    complex (C5b-C9) and quantification of complement split products by precipitation-in-gel techniques (e.g. C3d). We have developed a mouse monoclonal antibody (mAb) that is able to detect fluid phase C3c without interference from other products generated from the complement component C3. The C3c specific m......Ab was tested in different ELISA combinations with various types of in vitro activated sera and with plasma or serum samples from factor I deficient patients. The specificity of the mAb was evaluated in immunoprecipitation techniques and by analysis of eluted fragments of C3 after immunoaffinity...... chromatography. The C3c mAb was confirmed to be C3c specific, as it showed no cross-reactivity with native (un-cleaved) C3, with C3b, iC3b, or with C3d. Also, no significant reaction was observed with C3 fragments in factor I deficient sera or plasma. This antibody forms the basis for the generation of a robust...

  6. From galaxy-scale fueling to nuclear-scale feedback: the merger-state of radio galaxies 3C293, 3C305 & 4C12.50

    CERN Document Server

    Emonts, Bjorn; Villar-Martin, Montserrat; Hodgson, Jeff; Brogt, Erik; Tadhunter, Clive; Mahony, Elizabeth; Oosterloo, Tom

    2016-01-01

    Powerful radio galaxies are often associated with gas-rich galaxy mergers. These mergers may provide the fuel to trigger starburst and active galactic nuclear (AGN) activity. In this Research Note, we study the host galaxies of three seemingly young or re-started radio sources that drive fast outflows of cool neutral hydrogen (HI) gas, namely 3C 293, 3C 305 and 4C 12.50 (PKS 1345+12). Our aim is to link the feedback processes in the central kpc-scale region with new information on the distribution of stars and gas at scales of the galaxy. For this, we use deep optical V-band imaging of the host galaxies, complemented with HI emission-line observations to study their gaseous environments. We find prominent optical tidal features in all three radio galaxies, which confirm previous claims that 3C 293, 3C 305 and 4C 12.50 have been involved in a recent galaxy merger or interaction. Our data show the complex morphology of the host galaxies, and identify the companion galaxies that are likely involved in the merger...

  7. 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.

  8. Inhibitors of lysosomal cysteine proteases

    Directory of Open Access Journals (Sweden)

    Lyanna O. L.

    2011-04-01

    Full Text Available The review is devoted to the inhibitors of cysteine proteinases which are believed to be very important in many biochemical processes of living organisms. They participate in the development and progression of numerous diseases that involve abnormal protein turnover. One of the main regulators of these proteinases is their specific inhibitors: cystatins. The aim of this review was to present current knowledge about endogenous inhibitors of lysosomal cysteine proteases and their synthetic analogs.

  9. 3C 33: another case of photoionized soft X-ray emission in radio galaxies

    OpenAIRE

    Torresi, E.; Grandi, P; Guainazzi, M.; Palumbo, G. G. C.; Ponti, G; Bianchi, S.

    2009-01-01

    All the observations available in the Chandra and XMM-Newton archives have been used to investigate the X-ray spectral properties of 3C 33. In this paper is presented a complete X-ray analysis of the nuclear emission of this narrow line radio galaxy. The broad band spectrum of 3C 33 is complex. The hard part resembles that of Seyfert 2 galaxies, with a heavily obscured nuclear continuum (N_H~10^23 cm^-2) and a prominent Fe Kalpha line. This represents the nuclear radiation directly observed i...

  10. Privacy Issues of the W3C Geolocation API

    CERN Document Server

    Doty, Nick; Wilde, Erik

    2010-01-01

    The W3C's Geolocation API may rapidly standardize the transmission of location information on the Web, but, in dealing with such sensitive information, it also raises serious privacy concerns. We analyze the manner and extent to which the current W3C Geolocation API provides mechanisms to support privacy. We propose a privacy framework for the consideration of location information and use it to evaluate the W3C Geolocation API, both the specification and its use in the wild, and recommend some modifications to the API as a result of our analysis.

  11. Privacy Issues of the W3C Geolocation API

    OpenAIRE

    Doty, Nick; Mulligan, Deirdre K.; Wilde, Erik

    2010-01-01

    The W3C's Geolocation API may rapidly standardize the transmission of location information on the Web, but, in dealing with such sensitive information, it also raises serious privacy concerns. We analyze the manner and extent to which the current W3C Geolocation API provides mechanisms to support privacy. We propose a privacy framework for the consideration of location information and use it to evaluate the W3C Geolocation API, both the specification and its use in the wild, and recommend s...

  12. Molecular Gas in the Powerful Radio Galaxies 3C~31 and 3C~264 Major or Minor Mergers?

    CERN Document Server

    Lim, J; Combes, F

    2000-01-01

    We report the detection of $^{12}$CO~($1 \\to 0$) and $^{12}$CO~($2 \\to 1$) emission from the central regions ($\\lesssim 5$--$10 {\\rm kpc}$) of the two powerful radio galaxies 3C~31 and 3C~264. Their individual CO emission exhibits a double-horned line profile that is characteristic of an inclined rotating disk with a central depression at the rising part of its rotation curve. The inferred disk or ring distributions of the molecular gas is consistent with the observed presence of dust disks or rings detected optically in the cores of both galaxies. For a CO to H$_2$ conversion factor similar to that of our Galaxy, the corresponding total mass in molecular hydrogen gas is $(1.3 \\pm 0.2) \\times 10^9 {\\rm M_{\\odot}}$ in 3C~31 and $(0.31 \\pm 0.06) \\times 10^9 {\\rm M_{\\odot}}$ in 3C~264. Despite their relatively large molecular-gas masses and other peculiarities, both 3C~31 and 3C~264, as well as many other powerful radio galaxies in the (revised) 3C catalog, are known to lie within the fundamental plane of normal...

  13. Extracellular proteases as targets for drug development.

    Science.gov (United States)

    Cudic, Mare; Fields, Gregg B

    2009-08-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addition to directly blocking the activity of a targeted protease, one can take advantage of differential expression in disease states to selectively deliver therapeutic or imaging agents. Recent studies in targeted drug development for the metalloproteases (matrix metalloproteinases, adamalysins, pappalysins, neprilysin, angiotensin-converting enzyme, metallocarboxypeptidases, and glutamate carboxypeptidase II), serine proteases (elastase, coagulation factors, tissue/urokinase plasminogen activator system, kallikreins, tryptase, dipeptidyl peptidase IV) and cysteine proteases (cathepsin B) are discussed herein. PMID:19689354

  14. Optical Periodicity Analysis of 3C 446 using Period04

    Indian Academy of Sciences (India)

    Fei Guo; Hao Jing Zhang

    2014-09-01

    All the data of the blazar 3C446 at 8, 4.8, 14 and 22 GHz, presented in publications from 1977 to 2006, have been compiled to generate light curves. The light curves show violent activity of 3C446. Using Period04 analysis method, we have found that there is a period of 7.2 yr, which is consistent with the results that we found using wavelet analysis method. We get the instability region as = 123.83.

  15. Stimulation of Naive Monocytes and PBMCs with Coagulation Proteases Results in Thrombin-Mediated and PAR-1-Dependent Cytokine Release and Cell Proliferation in PBMCs Only

    NARCIS (Netherlands)

    Nieuwenhuizen, L.; Falkenburg, W. J. J.; Schutgens, R. E. G.; Roosendaal, G.; van Veghel, K.; Biesma, D. H.; Lafeber, F. P. J. G.

    2013-01-01

    Protease-activated receptors (PARs) are stimulated by proteolytic cleavage of their extracellular domain. Coagulation proteases, such as FVIIa, the binary TF-FVIIa complex, free FXa, the ternary TF-FVIIa-FXa complex and thrombin, are able to stimulate PARs. Whereas the role of PARs on platelets is w

  16. Orchestration of an uncommon maturation cascade of the house dust mite protease allergen quartet

    Directory of Open Access Journals (Sweden)

    Marie-Eve eDumez

    2014-03-01

    Full Text Available In more than 20% of the world population, sensitization to house dust mite (HDM allergens triggers typical allergic diseases such as allergic rhinitis and asthma. Amongst the 23 mite allergen groups hitherto identified, groups 1 are cysteine proteases belonging to the papain-like family whereas groups 3, 6 and 9 are serine proteases displaying trypsin, chymotrypsin and collagenolytic activities, respectively. While these proteases are more likely to be involved in the mite digestive system, they also play critical roles in the initiation and in the chronicity of the allergic response notably through the activation of innate immune pathways. All these allergenic proteases are expressed in mite as inactive precursor form. Until recently, the exact mechanisms of their maturation into active proteases remained to be fully elucidated. Recent breakthroughs in the understanding of the activation mechanisms of mite allergenic protease precursors have highlighted an uncommon and unique maturation pathway orchestrated by group 1 proteases that tightly regulates the proteolytic activities of groups 1, 3, 6 and 9 through complex intra- or intermolecular mechanisms. This review presents and discusses the currently available knowledge of the activation mechanisms of group 1, 3, 6 and 9 allergens of Dermatophagoides pteronyssinus laying special emphasis on their localization, regulation and interconnection.

  17. Discrimination of differentially inhibited cysteine proteases by activity-based profiling using cystatin variants with tailored specificities.

    Science.gov (United States)

    Sainsbury, Frank; Rhéaume, Ann-Julie; Goulet, Marie-Claire; Vorster, Juan; Michaud, Dominique

    2012-12-01

    Recent research has shown the possibility of tailoring the inhibitory specificity of plant cystatins toward cysteine (Cys) proteases by single mutations at positively selected amino acid sites. Here we devised a cystatin activity-based profiling approach to assess the impact of such mutations at the proteome scale using single variants of tomato cystatin SlCYS8 and digestive Cys proteases of the herbivorous insect, Colorado potato beetle, as a model. Biotinylated forms of SlCYS8 and SlCYS8 variants were used to capture susceptible Cys proteases in insect midgut protein extracts by biotin immobilization on avidin-embedded beads. A quantitative LC-MS/MS analysis of the captured proteins was performed to compare the inhibitory profile of different SlCYS8 variants. The approach confirmed the relevance of phylogenetic inferences categorizing the insect digestive Cys proteases into six functionally distinct families. It also revealed significant variation in protease family profiles captured with N-terminal variants of SlCYS8, in line with in silico structural models for Cys protease-SlCYS8 interactions suggesting a functional role for the N-terminal region. Our data confirm overall the usefulness of cystatin activity-based protease profiling for the monitoring of Cys protease-inhibitor interactions in complex biological systems. They also illustrate the potential of biotinylated cystatins to identify recombinant cystatin candidates for the inactivation of specific Cys protease targets.

  18. Extracellular proteases as targets for drug development

    OpenAIRE

    Cudic, Mare; Fields, Gregg B.

    2009-01-01

    Proteases constitute one of the primary targets in drug discovery. In the present review, we focus on extracellular proteases (ECPs) because of their differential expression in many pathophysiological processes, including cancer, cardiovascular conditions, and inflammatory, pulmonary, and periodontal diseases. Many new ECP inhibitors are currently under clinical investigation and a significant increase in new therapies based on protease inhibition can be expected in the coming years. In addit...

  19. A biotechnology perspective of fungal proteases

    OpenAIRE

    Paula Monteiro Souza; Mona Lisa de Assis Bittencourt; Carolina Canielles Caprara; Marcela de Freitas; Renata Paula Coppini de Almeida; Dâmaris Silveira; Yris Maria Fonseca; Edivaldo Ximenes Ferreira Filho; Adalberto Pessoa Junior; Pérola Oliveira Magalhães

    2015-01-01

    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...

  20. Nucleotide sequences encoding a thermostable alkaline protease

    Science.gov (United States)

    Wilson, David B.; Lao, Guifang

    1998-01-01

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium.

  1. Structure, electronic, mechanical and optical properties of ternary YAl3C3 carbide

    Science.gov (United States)

    Hussain, Altaf; Javed, Athar; Mehmood, Salman; Rasool, M. Nasir; Khan, Muhammad Azhar; Iqbal, Faisal

    2016-05-01

    The electronic structure, mechanical and optical properties of ternary yttrium aluminum carbide (YAl3C3) has been studied by first principles approach. The crystal structure and elastic properties are studied by using Vienna ab initio simulation package (VASP). An orthogonalized linear combination of atomic orbitals (OLCAO) method based on the density functional theory (DFT) is implemented to elucidate the electronic structure and optical properties of ternary YAl3C3 carbide. The YAl3C3 carbide exhibits a narrow indirect band gap, Eg=0.12 eV which shows its poor metallic and/or semiconductor behavior. The effective charge (Q*) calculation reveals more charge transfer from Al-sites as compared to Y-sites which indicates dominant ionic character of Al-sites. The analysis of structure and bond order (BO) calculations show that the Al-C bonds in the basal plane are much stronger as compared to Al-C bonds along the c-axis. The Al-C bonds lying in the basal plane have main contribution into the overall stiffness of YAl3C3 carbide. The effective mass of charge carriers (electrons and holes) and inter-band optical properties (complex dielectric function and optical conductivity) are also studied which show high degree of anisotropy in YAl3C3.

  2. Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

    Directory of Open Access Journals (Sweden)

    Sungho Ghil

    Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

  3. Complexity

    CERN Document Server

    Gershenson, Carlos

    2011-01-01

    The term complexity derives etymologically from the Latin plexus, which means interwoven. Intuitively, this implies that something complex is composed by elements that are difficult to separate. This difficulty arises from the relevant interactions that take place between components. This lack of separability is at odds with the classical scientific method - which has been used since the times of Galileo, Newton, Descartes, and Laplace - and has also influenced philosophy and engineering. In recent decades, the scientific study of complexity and complex systems has proposed a paradigm shift in science and philosophy, proposing novel methods that take into account relevant interactions.

  4. 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.

  5. Structural insights into the unique inhibitory mechanism of the silkworm protease inhibitor serpin18

    Science.gov (United States)

    Guo, Peng-Chao; Dong, Zhaoming; Zhao, Ping; Zhang, Yan; He, Huawei; Tan, Xiang; Zhang, Weiwei; Xia, Qingyou

    2015-01-01

    Serpins generally serve as inhibitors that utilize a mobile reactive center loop (RCL) as bait to trap protease targets. Here, we present the crystal structure of serpin18 from Bombyx mori at 1.65 Å resolution, which has a very short and stable RCL. Activity analysis showed that the inhibitory target of serpin18 is a cysteine protease rather than a serine protease. Notably, this inhibitiory reaction results from the formation of an intermediate complex, which then follows for the digestion of protease and inhibitor into small fragments. This activity differs from previously reported modes of inhibition for serpins. Our findings have thus provided novel structural insights into the unique inhibitory mechanism of serpin18. Furthermore, one physiological target of serpin18, fibroinase, was identified, which enables us to better define the potential role for serpin18 in regulating fibroinase activity during B. mori development. PMID:26148664

  6. C1A cysteine protease-cystatin interactions in leaf senescence.

    Science.gov (United States)

    Díaz-Mendoza, Mercedes; Velasco-Arroyo, Blanca; González-Melendi, Pablo; Martínez, Manuel; Díaz, Isabel

    2014-07-01

    Senescence-associated proteolysis in plants is a crucial process to relocalize nutrients from leaves to growing or storage tissues. The massive net degradation of proteins involves broad metabolic networks, different subcellular compartments, and several types of proteases and regulators. C1A cysteine proteases, grouped as cathepsin L-, B-, H-, and F-like according to their gene structures and phylogenetic relationships, are the most abundant enzymes responsible for the proteolytic activity during leaf senescence. Besides, cystatins as specific modulators of C1A peptidase activities exert a complex regulatory role in this physiological process. This overview article covers the most recent information on C1A proteases in leaf senescence in different plant species. Particularly, it is focussed on barley, as the unique species where the whole gene family members of C1A cysteine proteases and cystatins have been analysed.

  7. Epstein-Barr Virus Nuclear Antigen 3C Augments Mdm2-Mediated p53 Ubiquitination and Degradation by Deubiquitinating Mdm2▿

    OpenAIRE

    Saha, Abhik; Murakami, Masanao; Kumar, Pankaj; Bajaj, Bharat; Sims, Karen; Erle S Robertson

    2009-01-01

    Epstein-Barr virus (EBV) nuclear antigen 3C (EBNA3C) is one of the essential latent antigens for primary B-cell transformation. Previous studies established that EBNA3C facilitates degradation of several vital cell cycle regulators, including the retinoblastoma (pRb) and p27KIP proteins, by recruitment of the SCFSkp2 E3 ubiquitin ligase complex. EBNA3C was also shown to be ubiquitinated at its N-terminal residues. Furthermore, EBNA3C can bind to and be degraded in vitro by purified 20S protea...

  8. Suzaku Observations of the Radio Galaxy 3C 33

    CERN Document Server

    Evans, Daniel A; Hardcastle, Martin J; Kraft, Ralph P; Lee, Julia C; Virani, Shanil N

    2009-01-01

    We present results from a new 100-ks Suzaku observation of the nearby radio galaxy 3C 33, and investigate the nature of absorption, reflection, and jet production in this source. We model the 2-70 keV nuclear continuum with a power law that is absorbed either through one or more layers of pc-scale neutral material, or through a modestly ionized pc-scale obscurer. The expected signatures of reflection from a neutral accretion disk are absent in 3C 33: there is no evidence of a relativistically blurred Fe K$\\alpha$ emission line, and no Compton reflection hump above 10 keV. We discuss the implications of this for the nature of jet production in 3C 33.

  9. Immobilization to prevent enzyme incompatibility with proteases

    NARCIS (Netherlands)

    Vossenberg, P.; Beeftink, H.H.; Cohen Stuart, M.A.; Tramper, J.

    2011-01-01

    Enzyme incompatibility is a problem in multi-enzyme processes that involve a non-specific protease, such as Alcalase. An example is the one-pot enzymatic synthesis of peptides catalyzed by a lipase and a protease. The incompatibility between lipase B from Candida antarctica (CalB) and Alcalase was s

  10. 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...

  11. A microelectromechanical system digital 3C array seismic cone penetrometer

    OpenAIRE

    Ghose, R.

    2012-01-01

    A digital 3C array seismic cone penetrometer has been developed for multidisciplinary geophysical and geotechnical applications. Seven digital triaxial microelectromechanical system accelerometers are installed at 0.25-m intervals to make a 1.5-m-long downhole seismic array. The accelerometers have a flat response up to 2 kHz. The seismic array is attached to a class 1 digital seismic cone, which measures cone tip resistance, sleeve friction, pore-pressure, and inclination. The downhole 3C ar...

  12. Suzaku Observations of the Radio Galaxy 3C 33

    OpenAIRE

    Evans, A. Daniel; Reeves, James N.; Hardcastle, Martin J.; Kraft, Ralph P.; Lee, Julia C.; Virani, Shanil N.

    2010-01-01

    We present results from a new 100 - ks Suzaku observation of the nearby radio galaxy 3C 33, and investigate the nature of absorption, reflection, and jet production in this source. We model the 2 - 70 keV nuclear continuum with a power law that is absorbed either through one or more layers of pc-scale neutral material, or through a modestly ionized pc-scale obscurer. The expected signatures of reflection from a neutral accretion disk are absent in 3C 33 : there is no evidence of a relativisti...

  13. Suzaku Observations of the Radio Galaxy 3C 33

    OpenAIRE

    Evans, Daniel A.; Reeves, James N.; Hardcastle, Martin J.; Kraft, Ralph P.; Lee, Julia C.; Virani, Shanil N.

    2009-01-01

    We present results from a new 100-ks Suzaku observation of the nearby radio galaxy 3C 33, and investigate the nature of absorption, reflection, and jet production in this source. We model the 2-70 keV nuclear continuum with a power law that is absorbed either through one or more layers of pc-scale neutral material, or through a modestly ionized pc-scale obscurer. The expected signatures of reflection from a neutral accretion disk are absent in 3C 33: there is no evidence of a relativistically...

  14. New infrared spectral component of the quasar 3C273

    Energy Technology Data Exchange (ETDEWEB)

    Robson, E.I.; Gear, W.K.; Brown, L.M.J.; Courvoisier, T.J.-L.; Smith, M.G.; Griffin, M.J.; Blecha, A.

    1986-09-11

    Following the dramatic infrared to millimetre-wavelength flare seen in the quasar 3C273 during 1983, the authors have continued to monitor its overall continuum emission. Recent measurements show that the 10-..mu..m to 3-mm emission has decayed to a level well below any seen previously, while the 1-4-..mu..m emission has remained relatively constant. This behaviour has revealed the presence of an apparently non-variable component which dominates the near-infrared emission in 3C273 and includes the small 'bump' at approx. 3.5 ..mu..m in the power-law continuum.

  15. Peculiar variations in the structure of the quasar 3C454.3

    International Nuclear Information System (INIS)

    Following a major outburst at radio wavelengths in the quasar 3C454.3, observations by means of very long baseline interferometry have revealed a superluminal increase in the size of the compact 'core' region of the source and the appearance of a complex structure within it. Some features within this structure remained stationary with respect to each other, but others showed superluminal motion. This behaviour is distinctly different from that generally seen in superluminal sources. (author)

  16. Peculiar variations in the structure of the quasar 3C454. 3

    Energy Technology Data Exchange (ETDEWEB)

    Pauliny-Toth, I.I.K.; Porcas, R.W.; Zensus, J.A.; Kellermann, K.I.; Wu, S.Y.; Nicolson, G.D.; Mantovani, F.

    1987-08-27

    Following a major outburst at radio wavelengths in the quasar 3C454.3, observations by means of very long baseline interferometry have revealed a superluminal increase in the size of the compact 'core' region of the source and the appearance of a complex structure within it. Some features within this structure remained stationary with respect to each other, but others showed superluminal motion. This behaviour is distinctly different from that generally seen in superluminal sources.

  17. Progress and prospects on DENV protease inhibitors.

    Science.gov (United States)

    Timiri, Ajay Kumar; Sinha, Barij Nayan; Jayaprakash, Venkatesan

    2016-07-19

    New treatments are desperately required to combat increasing rate of dengue fever cases reported in tropical and sub-tropical parts of the world. Among the ten proteins (structural and non-structural) encoded by dengue viral genome, NS2B-NS3 protease is an ideal target for drug discovery. It is responsible for the processing of poly protein that is required for genome replication of the virus. Moreover, inhibitors designed against proteases were found successful in Human Immuno-deficiency Virus (HIV) and Hepatitis C Virus (HCV). Complete molecular mechanism and a survey of inhibitors reported against dengue protease will be helpful in designing effective and potent inhibitors. This review provides an insight on molecular mechanism of dengue virus protease and covers up-to-date information on different inhibitors reported against dengue proteases with medicinal chemistry perspective. PMID:27092412

  18. Protease-degradable electrospun fibrous hydrogels

    Science.gov (United States)

    Wade, Ryan J.; Bassin, Ethan J.; Rodell, Christopher B.; Burdick, Jason A.

    2015-03-01

    Electrospun nanofibres are promising in biomedical applications to replicate features of the natural extracellular matrix (ECM). However, nearly all electrospun scaffolds are either non-degradable or degrade hydrolytically, whereas natural ECM degrades proteolytically, often through matrix metalloproteinases. Here we synthesize reactive macromers that contain protease-cleavable and fluorescent peptides and are able to form both isotropic hydrogels and electrospun fibrous hydrogels through a photoinitiated polymerization. These biomimetic scaffolds are susceptible to protease-mediated cleavage in vitro in a protease dose-dependent manner and in vivo in a subcutaneous mouse model using transdermal fluorescent imaging to monitor degradation. Importantly, materials containing an alternate and non-protease-cleavable peptide sequence are stable in both in vitro and in vivo settings. To illustrate the specificity in degradation, scaffolds with mixed fibre populations support selective fibre degradation based on individual fibre degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications.

  19. 基于S3C2410上U-Boot的移植与实现%U-Boot’s Transplantation and Implementation Based on S3C2410

    Institute of Scientific and Technical Information of China (English)

    张伟; 刘斌; 董群锋

    2014-01-01

    The operating system kernel transplantation is the premise and foundation of the embedded system development. In view of the complexity and diversity of the U-boot transplantation, this paper analyzed the file structure and starting process of U-boot, it has chosen S3C2410 of SanSung company for development board, cross-compilation environment construction and U-boot transplantation and the kernel of the burning process were introduced in detail. We combined the function of U-boot with the characteristics of Linux in the transplantation process, this method has the characters of fast transplant, simple modify of kernel and strong commonality. Through compile testing, U-boot transplantation is implemented successful on the S3C2410, provides a reference for other U-boot transplantation.%操作系统内核移植是嵌入式系统开发的前提和基础,针对U-boot移植的复杂性和多样性,在分析了U-boot的文件结构和启动过程的基础上,选取了以SanSung公司的S3C2410为处理器的开发板,详细介绍了交叉编译环境的搭建、U-boot的移植、内核的烧写等过程。移植过程中将U-boot的功能与Linux的特点相结合,此方法具有移植速度快、内核修改简单、通用性强的特点。通过编译测试,成功实现了U-boot在S3C2410的移植,为其他U-boot的移植提供了一种参考。

  20. 基于S3C2410上U-Boot的移植与实现%U-Boot’s Transplantation and Implementation Based on S3C2410

    Institute of Scientific and Technical Information of China (English)

    张伟; 刘斌; 董群锋

    2014-01-01

    操作系统内核移植是嵌入式系统开发的前提和基础,针对U-boot移植的复杂性和多样性,在分析了U-boot的文件结构和启动过程的基础上,选取了以SanSung公司的S3C2410为处理器的开发板,详细介绍了交叉编译环境的搭建、U-boot的移植、内核的烧写等过程。移植过程中将U-boot的功能与Linux的特点相结合,此方法具有移植速度快、内核修改简单、通用性强的特点。通过编译测试,成功实现了U-boot在S3C2410的移植,为其他U-boot的移植提供了一种参考。%The operating system kernel transplantation is the premise and foundation of the embedded system development. In view of the complexity and diversity of the U-boot transplantation, this paper analyzed the file structure and starting process of U-boot, it has chosen S3C2410 of SanSung company for development board, cross-compilation environment construction and U-boot transplantation and the kernel of the burning process were introduced in detail. We combined the function of U-boot with the characteristics of Linux in the transplantation process, this method has the characters of fast transplant, simple modify of kernel and strong commonality. Through compile testing, U-boot transplantation is implemented successful on the S3C2410, provides a reference for other U-boot transplantation.

  1. W3C head Berners-Lee to be knighted

    CERN Multimedia

    Gross, G

    2004-01-01

    "Tim Berners-Lee, credited with inventing the World Wide Web and now director of the World Wide Web Consortium, will be named a knight commander, Order of the British Empire, by Queen Elizabeth II, the W3C announced Wednesday" (1 page)

  2. Search for exotic events from the L3+C data

    Institute of Scientific and Technical Information of China (English)

    DING Lin-Kai; HE Zuo-Xiu; HUO An-Xiang; JING Cai-Liu; KUANG Hao-Huai; LEI Yu; LI Li; MA Xin-Hua; MA Yu-Qian; QING Cheng-Rui; WANG Rui-Guang; YAO Zhi-Guo; YU Zhong-Qiang; ZHANG Chao; ZHANG Feng; ZHANG Jing; ZHU Qing-Qi

    2009-01-01

    An effort to search for Kolar-like events within the data set of the L3+C experiment is reported. From a total of 0.89×1010 triggered events there are no reliable two-prong Kolar-like events observed. The some reasonable assumptions.

  3. The Double–Double Radio Galaxy 3C293

    Indian Academy of Sciences (India)

    S. A. Joshi; S. Nandi; D. J. Saikia; C. H. Ishwara-Chandra; C. Konar

    2011-12-01

    We present the results of radio continuum observations at frequencies ranging from ∼ 150–5000 MHz of the misaligned double–double radio galaxy (DDRG) 3C293 (J1352+3126) using the GMRT and the VLA, and estimate the time-scale of interruption of jet activity to be less than ∼ 0.1 Myr.

  4. Position Measurements of the Core in 3C 66B

    Indian Academy of Sciences (India)

    G.-Y. Zhao; Y.-J. Chen; Z.-Q. Shen; H. Sudou; S. Iguchi; Y. Murata; Y. Taniguchi

    2011-03-01

    It was argued that 3C 66B, a nearby radio galaxy, harbors a supermassive black hole binary (SMBHB). To investigate this, a 4-epoch VLBA phase referencing imaging observation was performed in 2004–2005. Here we present some preliminary results of this project. We found a large position difference compared to previous results.

  5. A 3C-path for Glauberman-Norton theory

    CERN Document Server

    Griess, Robert L

    2009-01-01

    One would like an explanation of the provocative McKay and Glauberman-Norton observations connecting the extended $E_8$-diagram with pairs of 2A involutions in the Monster sporadic simple group. We propose a down-to-earth model for the 3C-case which exhibits a logic to these connections.

  6. Revisiting correlations between broad-line and jet emission variations for AGNs: 3C 120 and 3C 273

    Science.gov (United States)

    Liu, H. T.; Bai, J. M.; Feng, H. C.; Li, S. K.

    2015-06-01

    We restudy the issue of cross-correlations between broad-line and jet emission variations, and aim to locate the position of a radio (and gamma-ray) emitting region in a jet of active galactic nuclei. Considering the radial profiles of the radius and number density of clouds in a spherical broad-line region (BLR), we derive new formulae connecting the jet-emitting position Rjet to the time lag τob between broad-line and jet emission variations, and the BLR radius. Also, formulae are derived for a disc-like BLR and a spherical shell BLR. The model-independent flux randomization/random subset selection method is used to estimate τob. For 3C 120, positive lags of about 0.3 yr are found between the 15 GHz emission and the Hβ, Hγ and He II λ4686 lines, including broad-line data in a newly published paper, indicating that the line variations lead the 15 GHz ones. Each of the broad-line light curves corresponds to a radio outburst. Rjet = 1.1-1.5 parsec (pc) is obtained for 3C 120. For 3C 273, a common feature of negative time lags is found in the cross-correlation functions between light curves of radio emission and the Balmer lines, as well as Lyα λ1216 and C IV λ1549 lines. Rjet = 1.0-2.6 pc is obtained for 3C 273. The estimated Rjet is comparable for 3C 120 and 3C 273, and the gamma-ray-emitting positions will be within ˜1-3 pc from the central engines. Comparisons show that the cloud number density and radius radial distributions and the BLR structures have only negligible effects on Rjet.

  7. Connection Between X-Ray Emission and Relativistic Jets in the Radio Galaxies 3C 111 and 3C 120

    Science.gov (United States)

    Aller, Margo F.

    2005-01-01

    This work represents a part of a longterm study of the X-ray flux variability in radio galaxies and its relation to flux and structural changes in the associated radio jet. The work described here included: 1) continued study of the emission properties of the FR I radio galaxy 3C 120 known to exhibit a jet/disk connection from our past work; and 2) the commencement of monitoring of a second radio galaxy, the FR I1 object 3C 111 which was selected because of similar radio and X-ray properties to 3C 120, including the presence of Fe K a emission. The association between X-ray dips and new superluminal components, suggesting a picture in which the radio jet is fed by accretion events near the black hole, was identified in 3C 120 using combined RXTE and radio flux monitoring data and bi-monthly to monthly imaging data from the VLBA at 43 GHz. Such data were also obtained for both targets during the period described here. Specific goals were to more broadly investigate the X-ray dip/superluminal connection in 3C 120, thereby determining the epochs of X-ray minima and superluminal ejections more accurately (and hence more precisely determining the distance between the accretion disk and the core of the radio jet), and to determine whether a similar pattern is present in the data for a second radio galaxy. In 3C 111 a different time scale (longer time delays between X-ray dips and superluminal ejections) was expected due to the higher black hole mass implied by its higher radio luminosity: no black hole mass is published for this object but one can be determined from a PDS analysis of the RXTE data. The addition of the second source to the study would identify whether a similar connection was present in other sources and, if found, would provide important information on how time scale (and hence size scale) of accretion disk/jet systems depends on black hole mass. The grant included funding for the reduction and analysis of data obtained during the time period of Rossi

  8. Evolutionary implications of C3 -C4 intermediates in the grass Alloteropsis semialata.

    Science.gov (United States)

    Lundgren, Marjorie R; Christin, Pascal-Antoine; Escobar, Emmanuel Gonzalez; Ripley, Brad S; Besnard, Guillaume; Long, Christine M; Hattersley, Paul W; Ellis, Roger P; Leegood, Richard C; Osborne, Colin P

    2016-09-01

    C4 photosynthesis is a complex trait resulting from a series of anatomical and biochemical modifications to the ancestral C3 pathway. It is thought to evolve in a stepwise manner, creating intermediates with different combinations of C4 -like components. Determining the adaptive value of these components is key to understanding how C4 photosynthesis can gradually assemble through natural selection. Here, we decompose the photosynthetic phenotypes of numerous individuals of the grass Alloteropsis semialata, the only species known to include both C3 and C4 genotypes. Analyses of δ(13) C, physiology and leaf anatomy demonstrate for the first time the existence of physiological C3 -C4 intermediate individuals in the species. Based on previous phylogenetic analyses, the C3 -C4 individuals are not hybrids between the C3 and C4 genotypes analysed, but instead belong to a distinct genetic lineage, and might have given rise to C4 descendants. C3 A. semialata, present in colder climates, likely represents a reversal from a C3 -C4 intermediate state, indicating that, unlike C4 photosynthesis, evolution of the C3 -C4 phenotype is not irreversible. PMID:26524631

  9. Caspase Family Proteases and Apoptosis

    Institute of Scientific and Technical Information of China (English)

    Ting-Jun FAN; Li-Hui HAN; Ri-Shan CONG; Jin LIANG

    2005-01-01

    Apoptosis, or programmed cell death, is an essential physiological process that plays a critical role in development and tissue homeostasis. The progress of apoptosis is regulated in an orderly way by a series of signal cascades under certain circumstances. The caspase-cascade system plays vital roles in the induction, transduction and amplification of intracellular apoptotic signals. Caspases, closely associated with apoptosis, are aspartate-specific cysteine proteases and members of the interleukin-1β-converting enzyme family. The activation and function of caspases, involved in the delicate caspase-cascade system, are regulated by various kinds of molecules, such as the inhibitor of apoptosis protein, Bcl-2 family proteins, calpain,and Ca2+. Based on the latest research, the members of the caspase family, caspase-cascade system and caspase-regulating molecules involved in apoptosis are reviewed.

  10. Structures of HIV Protease Guide Inhibitor Design to Overcome Drug Resistance

    Energy Technology Data Exchange (ETDEWEB)

    Weber, Irene T.; Kovalevsky, Andrey Y.; Harrison, Robert W. (GSU)

    2008-06-03

    The HIV/AIDS infection continues to be a major epidemic worldwide despite the initial promise of antiviral drugs. Current therapy includes a combination of drugs that inhibit two of the virally-encoded enzymes, the reverse transcriptase and the protease. The first generation of HIV protease inhibitors that have been in clinical use for treatment of AIDS since 1995 was developed with the aid of structural analysis of protease-inhibitor complexes. These drugs were successful in improving the life span of HIV-infected people. Subsequently, the rapid emergence of drug resistance has necessitated the design of new inhibitors that target mutant proteases. This second generation of antiviral protease inhibitors has been developed with the aid of data from medicinal chemistry, kinetics, and X-ray crystallographic analysis. Traditional computational methods such as molecular mechanics and dynamics can be supplemented with intelligent data mining approaches. One approach, based on similarities to the protease interactions with substrates, is to incorporate additional interactions with main chain atoms that cannot easily be eliminated by mutations. Our structural and inhibition data for darunavir have helped to understand its antiviral activity and effectiveness on drug resistant HIV and demonstrate the success of this approach.

  11. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors

    Directory of Open Access Journals (Sweden)

    Jesús Serrano-Luna

    2013-01-01

    Full Text Available The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.

  12. Proteases from Entamoeba spp. and Pathogenic Free-Living Amoebae as Virulence Factors.

    Science.gov (United States)

    Serrano-Luna, Jesús; Piña-Vázquez, Carolina; Reyes-López, Magda; Ortiz-Estrada, Guillermo; de la Garza, Mireya

    2013-01-01

    The standard reference for pathogenic and nonpathogenic amoebae is the human parasite Entamoeba histolytica; a direct correlation between virulence and protease expression has been demonstrated for this amoeba. Traditionally, proteases are considered virulence factors, including those that produce cytopathic effects in the host or that have been implicated in manipulating the immune response. Here, we expand the scope to other amoebae, including less-pathogenic Entamoeba species and highly pathogenic free-living amoebae. In this paper, proteases that affect mucin, extracellular matrix, immune system components, and diverse tissues and cells are included, based on studies in amoebic cultures and animal models. We also include proteases used by amoebae to degrade iron-containing proteins because iron scavenger capacity is currently considered a virulence factor for pathogens. In addition, proteases that have a role in adhesion and encystation, which are essential for establishing and transmitting infection, are discussed. The study of proteases and their specific inhibitors is relevant to the search for new therapeutic targets and to increase the power of drugs used to treat the diseases caused by these complex microorganisms.

  13. (Processing and targeting of the thiol protease aleurain)

    Energy Technology Data Exchange (ETDEWEB)

    Rogers, J.C.

    1990-01-01

    Our goal for work during the past two years under this Grant was to characterize the barley thiol protease, aleurain, to determine if it is secreted or retained intracellularly in aleurone cells, and to begin to elucidate structural features that might control targeting of the protein to its final destination. We have shown that aleurain is synthesized as a proenzyme with two N-linked oligosaccharide chains, one high mannose-type and one complex-type. Aleurain undergoes processing to mature form by removal of an Nterminal prosegment, and is retained intracellularly; it cannot be detected among proteins secreted from aleurone cells. Treatment of aleurone cells with tunicamycin to prevent glycosylation of aleurain does not prevent processing of the unglycosylated form. The N-terminal portion of aleurain's prosegment is homologous to the comparable region in two yeast vacuolar proteases, where that region is known to contain the signal necessary for targeting the proteases to the vacuole. 18 refs., 7 figs.

  14. Synthesis of C3/C1-Substituted Tetrahydroisoquinolines

    Directory of Open Access Journals (Sweden)

    Mohamed Mihoubi

    2015-08-01

    Full Text Available A broad biological screening of the natural alkaloid N-methylisosalsoline (2 extracted from Hammada scoparia leaves against a panel of human and parasitic proteases revealed an interesting activity profile of 2 towards human 20S proteasome. This outcome suggests that the 1,2,3,4-tetrahydroisoquinoline skeleton may be exploited as a template for the development of novel anticancer agents. In this article, we report the synthesis and chemical characterization of a new series of isosalsoline-type alkaloids (10–11 with variations at N2 and C3 positions with respect to the natural Compound 2, obtained by a synthetic strategy that involves the Bischler-Napieralski cyclization. The substrate for the condensation to the tetrahydroisoquinoline system, i.e., a functionalized β-arylethyl amine, was obtained through an original double reduction of nitroalkene. The synthetic strategy can be directed to the construction of highly substituted and functionalized 1,2,3,4-tetrahydroisoquinolines.

  15. Synthesis of C3/C1-Substituted Tetrahydroisoquinolines.

    Science.gov (United States)

    Mihoubi, Mohamed; Micale, Nicola; Scala, Angela; Jarraya, Raoudha Mezghani; Bouaziz, Amira; Schirmeister, Tanja; Risitano, Francesco; Piperno, Anna; Grassi, Giovanni

    2015-01-01

    A broad biological screening of the natural alkaloid N-methylisosalsoline (2) extracted from Hammada scoparia leaves against a panel of human and parasitic proteases revealed an interesting activity profile of 2 towards human 20S proteasome. This outcome suggests that the 1,2,3,4-tetrahydroisoquinoline skeleton may be exploited as a template for the development of novel anticancer agents. In this article, we report the synthesis and chemical characterization of a new series of isosalsoline-type alkaloids (10-11) with variations at N2 and C3 positions with respect to the natural Compound 2, obtained by a synthetic strategy that involves the Bischler-Napieralski cyclization. The substrate for the condensation to the tetrahydroisoquinoline system, i.e., a functionalized β-arylethyl amine, was obtained through an original double reduction of nitroalkene. The synthetic strategy can be directed to the construction of highly substituted and functionalized 1,2,3,4-tetrahydroisoquinolines. PMID:26287146

  16. Evolutionary analysis of novel serine proteases in the venom gland transcriptome of Bitis gabonica rhinoceros.

    Directory of Open Access Journals (Sweden)

    Sakthivel Vaiyapuri

    Full Text Available BACKGROUND: Serine proteases are major components of viper venom and target various stages of the blood coagulation system in victims and prey. A better understanding of the diversity of serine proteases and other enzymes present in snake venom will help to understand how the complexity of snake venom has evolved and will aid the development of novel therapeutics for treating snake bites. METHODOLOGY AND PRINCIPAL FINDINGS: Four serine protease-encoding genes from the venom gland transcriptome of Bitis gabonica rhinoceros were amplified and sequenced. Mass spectrometry suggests the four enzymes corresponding to these genes are present in the venom of B. g. rhinoceros. Two of the enzymes, rhinocerases 2 and 3 have substitutions to two of the serine protease catalytic triad residues and are thus unlikely to be catalytically active, though they may have evolved other toxic functions. The other two enzymes, rhinocerases 4 and 5, have classical serine protease catalytic triad residues and thus are likely to be catalytically active, however they have glycine rather than the more typical aspartic acid at the base of the primary specificity pocket (position 189. Based on a detailed analysis of these sequences we suggest that alternative splicing together with individual amino acid mutations may have been involved in their evolution. Changes within amino acid segments which were previously proposed to undergo accelerated change in venom serine proteases have also been observed. CONCLUSIONS AND SIGNIFICANCE: Our study provides further insight into the diversity of serine protease isoforms present within snake venom and discusses their possible functions and how they may have evolved. These multiple serine protease isoforms with different substrate specificities may enhance the envenomation effects and help the snake to adapt to new habitats and diets. Our findings have potential for helping the future development of improved therapeutics for snake bites.

  17. BeppoSAX Observations of 3C 279

    International Nuclear Information System (INIS)

    We report on BeppoSAX AO1 Core Program observations of 3C 279, performed in January 1997. 3C 279 was found in a low state, with constant X-ray flux in the 5 observations. The spectra obtained with the LECS and MECS instruments combining the 5 observations are well fitted by a single power law with energy spectral index α = 0.64 ± 0.03 and Galactic absorption. The source is weakly detected by the PDS instrument. Comparison with simultaneous γ-ray data obtained by EGRET and with previous multifrequency measurements shows that the X-ray emission is well correlated with the γ-ray emission over long timescales

  18. Insights into the serine protease mechanism based on structural observations of the conversion of a peptidyl serine protease inhibitor to a substrate

    DEFF Research Database (Denmark)

    Jiang, Longguang; Andersen, Lisbeth Moreau; Andreasen, Peter A;

    2016-01-01

    BACKGROUND: Serine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1) of a ser......BACKGROUND: Serine proteases are one of the most studied group of enzymes. Despite the extensive mechanistic studies, some crucial details remain controversial, for example, how the cleaved product is released in the catalysis reaction. A cyclic peptidyl inhibitor (CSWRGLENHRMC, upain-1......) of a serine protease, urokinase-type plasminogen activator (uPA), was found to become a slow substrate and cleaved slowly upon the replacement of single residue (W3A). METHODS: By taking advantage of the unique property of this peptide, we report the high-resolution structures of uPA in complex with upain-1-W...

  19. A Dust Lane in the Radio galaxy 3C270

    OpenAIRE

    Mahabal, Ashish; Kembhavi, Ajit; Singh, K. P.; Bhat, P.N.; Prabhu, T. P.

    1995-01-01

    We present broad band surface photometry of the radio galaxy 3C270 (NGC~4261). We find a distinct dust lane in the $V-R$ image of the galaxy, and determine its orientation and size. We use the major axis profile of the galaxy to estimate the optical depth of the dust lane, and discuss the significance of the lane to the shape of the galaxy.

  20. Pion elastic scattering from polarized sup 1 sup 3 C

    CERN Document Server

    Lee, D H; Kim, B T

    1999-01-01

    The elastic scattering cross sections and the analyzing powers for pi sup + and pi sup - from a sup 1 sup 3 C target are calculated by solving the Schroedinger equation reduced from the Klein-Gordon equation. In doing so, kinematical variables are redefined, local potentials are assumed , and the potential parameters are fixed to reproduce the experimental data. The calculated cross sections and the analyzing powers are compared with the observed data.

  1. A warm hot intergalactic medium towards 3C 120?

    CERN Document Server

    McKernan, B; Mushotzky, R; George, I M; Turner, T J

    2003-01-01

    We observed the Seyfert I active galaxy/broad line radio galaxy 3C120 with the Chandra high energy transmission gratings and present an analysis of the soft X-ray spectrum. We identify the strongest absorption feature (detected at >99.9% confidence) with O VIII Lya (FWHM=1010^{+295}_{-265} km/s), blueshifted by -5500 +/- 140 km/s from systemic velocity. The absorption may be due to missing baryons in warm/hot intergalactic medium (WHIGM) along the line-of-sight to 3C 120 at z=0.0147 +/- 0.0005, or it could be intrinsic to the jet of 3C 120. Assuming metallicities of 0.1 solar, we estimate an ionic column density of N_{O VIII}>3.4 \\times 10^{16} cm^{-2} for WHIGM and a filament depth of 56 relative to the critical density of a $\\Lambda$-dominated cold dark matter universe, which is in reasonable agreement with WHIGM simulations. We detect, at marginal significance, absorption of O VIII Lya at z\\sim 0 due to a hot medium in the Local Group. We also detect an unidentified absorption feature at \\sim 0.71 keV. Abs...

  2. The inner kiloparsec of the jet in 3C264

    CERN Document Server

    Lara, L; Cotton, B; Feretti, L; Venturi, T; Lara, Lucas; Giovannini, Gabriele; Cotton, Bill; Feretti, Luigina; Venturi, Tiziana

    2003-01-01

    We present new multi-frequency EVN, MERLIN and VLA observations of the radio source 3C264, sensitive to linear scales ranging from the parsec to several kiloparsecs. The observations confirm the existence of regions with different properties in the first kiloparsec of the jet. The most remarkable feature is the transition between a well collimated narrow jet at distances from the core below 80 pc, to a conical-shaped wide jet, with an opening angle of 20 degrees. Another change of properties, consisting of an apparent deflection of the jet ridge line and a diminution of the surface brightness, occurs at a distance of 300 pc from the core, coincident with the radius of a ring observed at optical wavelengths. Our observations add new pieces of information on the spectrum of the radio-optical jet of 3C264, with results consistent with a synchrotron emission mechanism and a spectrum break frequency in the infrared. Brightness profiles taken perpendicularly to the jet of 3C264 are consistent with a spine brightene...

  3. Dysregulation of Protease and Protease Inhibitors in a Mouse Model of Human Pelvic Organ Prolapse

    OpenAIRE

    Madhusudhan Budatha; Simone Silva; Teodoro Ignacio Montoya; Ayako Suzuki; Sheena Shah-Simpson; Cecilia Karin Wieslander; Masashi Yanagisawa; Ruth Ann Word; Hiromi Yanagisawa

    2013-01-01

    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 tryp...

  4. Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria

    Science.gov (United States)

    Goupil, Louise S.; Ivry, Sam L.; Hsieh, Ivy; Suzuki, Brian M.; Craik, Charles S.; O’Donoghue, Anthony J.; McKerrow, James H.

    2016-01-01

    Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites. PMID:27501047

  5. Cysteine and Aspartyl Proteases Contribute to Protein Digestion in the Gut of Freshwater Planaria.

    Science.gov (United States)

    Goupil, Louise S; Ivry, Sam L; Hsieh, Ivy; Suzuki, Brian M; Craik, Charles S; O'Donoghue, Anthony J; McKerrow, James H

    2016-08-01

    Proteases perform numerous vital functions in flatworms, many of which are likely to be conserved throughout the phylum Platyhelminthes. Within this phylum are several parasitic worms that are often poorly characterized due to their complex life-cycles and lack of responsiveness to genetic manipulation. The flatworm Schmidtea mediterranea, or planaria, is an ideal model organism to study the complex role of protein digestion due to its simple life cycle and amenability to techniques like RNA interference (RNAi). In this study, we were interested in deconvoluting the digestive protease system that exists in the planarian gut. To do this, we developed an alcohol-induced regurgitation technique to enrich for the gut enzymes in S. mediterranea. Using a panel of fluorescent substrates, we show that this treatment produces a sharp increase in proteolytic activity. These enzymes have broad yet diverse substrate specificity profiles. Proteomic analysis of the gut contents revealed the presence of cysteine and metallo-proteases. However, treatment with class-specific inhibitors showed that aspartyl and cysteine proteases are responsible for the majority of protein digestion. Specific RNAi knockdown of the cathepsin B-like cysteine protease (SmedCB) reduced protein degradation in vivo. Immunohistochemistry and whole-mount in situ hybridization (WISH) confirmed that the full-length and active forms of SmedCB are found in secretory cells surrounding the planaria intestinal lumen. Finally, we show that the knockdown of SmedCB reduces the speed of tissue regeneration. Defining the roles of proteases in planaria can provide insight to functions of conserved proteases in parasitic flatworms, potentially uncovering drug targets in parasites. PMID:27501047

  6. Solid-state characterization of the HIV protease inhibitor

    CERN Document Server

    Kim, Y A

    2002-01-01

    The LB71350, (3S, 4R)-Epoxy-(5S)-[[N-(1-methylethoxy) carbonyl]-3-(methylsulfonyl)-L-valinyl]amin= o]-N-[2-methyl-(1R)-[(phenyl)carbonyl]propyl-6-phenylhexanamide, is a novel HIV protease inhibitor. Its equilibrium solubility at room temperature was less than 40 mu g/mL. It was speculated that the low aqueous solubility might be due to the high crystalline lattice energy resulting from intermolecular hydrogen bonds. The present study was carried out to learn the solid-state characteristics of LB71350 using analytical methods such as NMR, FT-IR and XRD. sup 1 sup 3 C Solid-state NMR, solution NMR, and FT-IR spectra of the various solid forms of LB71350 were used to identify the conformation and structure of the solid forms. The chemical shifts of sup 1 sup 3 C solid-state NMR spectra suggest that the crystalline form might have 3 intermolecular hydrogen bondings between monomers.

  7. Production of Protease Enzyme from Wheat Straw

    Directory of Open Access Journals (Sweden)

    Mohammed A. Atiya

    2008-01-01

    Full Text Available Protease enzyme production was studied and optimized as a first step to collect information about solid state fermenter to produce protease enzyme. A local isolated Aspergillus niger was used for this study with constant spores feeding in every experiment at (105/g. Experiments carried out in conical flasks with (250 ml containing (10 g of wheat straw as a substrate with different conditions included temperature, pH, hydration ratio, and fermentation time, the results comprised by measuring protease activity (u. The results showed that the best activity can be obtained at (T = 32°C, t= 100 hrs, pH= 2.5 and hydration ratio is 1:3. On the other hand the results is courage to proceed to design a solid state protease fermenter from wheat straw.

  8. Vanadium inhibition of serine and cysteine proteases.

    Science.gov (United States)

    Guerrieri, N; Cerletti, P; De Vincentiis, M; Salvati, A; Scippa, S

    1999-03-01

    A study was made on the effect of vanadium, in both the tetravalent state in vanadyl sulphate and in the pentavalent state in sodium meta-vanadate, and ortho-vanadate, on the proteolysis of azocasein by two serine proteases, trypsin and subtilisin and two cysteine proteases bromelain and papain. Also the proteolysis of bovine azoalbumin by serine proteases was considered. An inhibitory effect was present in all cases, except meta-vanadate with subtilisin. The oxidation level of vanadium by itself did not determine the inhibition kinetics, which also depended on the type and composition of the vanadium containing molecule and on the enzyme assayed. The pattern of inhibition was similar for proteases belonging to the same class. The highest inhibition was obtained with meta-vanadate on papain and with vanadyl sulphate on bromelain.

  9. 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....

  10. 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...... for the extraction of lipases and proteases from activated sludge. The sludge was continuously stirred in the presence of either buffer alone or in the presence of detergent and/or chelating agents. In all cases, a marked reduction in floc size was observed upon continuous stirring. However, no lipase activity...... and negligible protease activity was extracted in the presence of buffer alone, indicating that enzyme extraction was not due to shear force alone. The highest lipase activity was extracted using 0.1% Triton X-100 above which the activity was gradually decreasing. For proteases, the highest activity was obtained...

  11. Electrically sensing protease activity with nanopores

    Science.gov (United States)

    Kukwikila, Mikiembo; Howorka, Stefan

    2010-11-01

    The enzymatic activity of a protease was electrically detected using nanopore recordings. A peptide substrate was tethered to microscale beads, and cleavage by the enzyme trypsin released a soluble fragment that was electrophoretically driven through the α-hemolysin protein pore, leading to detectable blockades in the ionic current. Owing to its simplicity, this approach to sense enzymatic activity may be applied to other proteases.

  12. Production of Protease Enzyme from Wheat Straw

    OpenAIRE

    Mohammed A. Atiya

    2008-01-01

    Protease enzyme production was studied and optimized as a first step to collect information about solid state fermenter) to produce protease enzyme. A local isolated Aspergillus niger was used for this study with constant spores feeding in every experiment at (105/g). Experiments carried out in conical flasks with (250 ml) containing (10 g) of wheat straw as a substrate with different conditions included temperature, pH, hydration ratio, and fermentation time, the results comprised by measuri...

  13. Neutral serine proteases of neutrophils.

    Science.gov (United States)

    Kettritz, Ralph

    2016-09-01

    Neutrophil serine proteases (NSPs) exercise tissue-degrading and microbial-killing effects. The spectrum of NSP-mediated functions grows continuously, not least because of methodological progress. Sensitive and specific FRET substrates were developed to study the proteolytic activity of each NSP member. Advanced biochemical methods are beginning to characterize common and specific NSP substrates. The resulting novel information indicates that NSPs contribute not only to genuine inflammatory neutrophil functions but also to autoimmunity, metabolic conditions, and cancer. Tight regulatory mechanisms control the proteolytic potential of NSPs. However, not all NSP functions depend on their enzymatic activity. Proteinase-3 (PR3) is somewhat unique among the NSPs for PR3 functions as an autoantigen. Patients with small-vessel vasculitis develop autoantibodies to PR3 that bind their target antigens on the neutrophil surface and trigger neutrophil activation. These activated cells subsequently contribute to vascular necrosis with life-threatening multiorgan failure. This article discusses various aspects of NSP biology and highlights translational aspects with strong clinical implications. PMID:27558338

  14. ADAM Proteases and Gastrointestinal Function.

    Science.gov (United States)

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis.

  15. ADAM Proteases and Gastrointestinal Function

    Science.gov (United States)

    Jones, Jennifer C.; Rustagi, Shelly; Dempsey, Peter J.

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  16. ADAM Proteases and Gastrointestinal Function.

    Science.gov (United States)

    Jones, Jennifer C; Rustagi, Shelly; Dempsey, Peter J

    2016-01-01

    A disintegrin and metalloproteinases (ADAMs) are a family of cell surface proteases that regulate diverse cellular functions, including cell adhesion, migration, cellular signaling, and proteolysis. Proteolytically active ADAMs are responsible for ectodomain shedding of membrane-associated proteins. ADAMs rapidly modulate key cell signaling pathways in response to changes in the extracellular environment (e.g., inflammation) and play a central role in coordinating intercellular communication within the local microenvironment. ADAM10 and ADAM17 are the most studied members of the ADAM family in the gastrointestinal tract. ADAMs regulate many cellular processes associated with intestinal development, cell fate specification, and the maintenance of intestinal stem cell/progenitor populations. Several signaling pathway molecules that undergo ectodomain shedding by ADAMs [e.g., ligands and receptors from epidermal growth factor receptor (EGFR)/ErbB and tumor necrosis factor α (TNFα) receptor (TNFR) families] help drive and control intestinal inflammation and injury/repair responses. Dysregulation of these processes through aberrant ADAM expression or sustained ADAM activity is linked to chronic inflammation, inflammation-associated cancer, and tumorigenesis. PMID:26667078

  17. 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.

  18. Delta-function Approximation SSC Model in 3C 273

    Indian Academy of Sciences (India)

    S. J. Kang; Y. G. Zheng; Q. Wu

    2014-09-01

    We obtain an approximate analytical solution using approximate calculation on the traditional one-zone synchrotron self-Compton (SSC) model. In this model, we describe the electron energy distribution by a broken power-law function with a sharp cut-off, and non-thermal photons are produced by both synchrotron and inverse Compton scattering of synchrotron photons. We calculate the radiation energy spectrum of electrons by the function. We apply this model to the multi-wavelength Spectral Energy Distributions (SED) of the 3C 273 in different states, and obtain excellent fits to the observed spectra of this source.

  19. Peculiar radio structure in the quasar 3C380

    Energy Technology Data Exchange (ETDEWEB)

    Wilkinson, P.N.; Booth, R.S.; Cornwell, T.J.; Clark, R.R.

    1984-04-12

    The authors present radio maps which hint at an explanation for the small size of at least some of the steep-spectrum compact sources (SSCSs). The maps, of the quasar 3C380, reveal a peculiar radio structure which is difficult to interpret other than by a powerful interaction between the radio-emitting plasma and its environment. Thus, the sub-galactic (projected) dimensions of this and other SSCSs may be a result of the beams being disrupted before they have propagated beyond the parent object.

  20. Peculiar radio structure in the quasar 3C380

    International Nuclear Information System (INIS)

    The authors present radio maps which hint at an explanation for the small size of at least some of the steep-spectrum compact sources (SSCSs). The maps, of the quasar 3C380, reveal a peculiar radio structure which is difficult to interpret other than by a powerful interaction between the radio-emitting plasma and its environment. Thus, the sub-galactic (projected) dimensions of this and other SSCSs may be a result of the beams being disrupted before they have propagated beyond the parent object. (author)

  1. Experience with Server Self Service Center (S3C)

    CERN Multimedia

    Sucik, J

    2009-01-01

    CERN has a successful experience with running Server Self Service Center (S3C) for virtual server provisioning which is based on Microsoft® Virtual Server 2005. With the introduction of Windows Server 2008 and its built-in hypervisor based virtualization (Hyper-V) there are new possibilities for the expansion of the current service. This paper describes the architecture of the redesigned virtual Server Self Service based on Hyper-V which provides dynamically scalable virtualized resources on demand as needed and outlines the possible implications on the future use of virtual machines at CERN.

  2. First results from the L3+C experiment at CERN

    CERN Document Server

    Ladrón de Guevara, P

    2001-01-01

    The L3+C experiment combines the high precision spectrometer of the L3 detector at LEP, CERN, with a small air shower array. The momenta of the cosmic ray induced muons can be measured from 20 to 2000 GeV /c. During the 1999 data taking period 5 billion muon events were recorded in the spectrometer. From April to November, 2000, an additional 6.8 billion muon events have been recorded as well as 33 million air shower events. Here the first results on the muon momentum spectrum and charge ratio will be presented. (9 refs).

  3. Experience with Server Self Service Center (S3C)

    CERN Document Server

    Sucik, J; CERN. Geneva. IT Department

    2010-01-01

    CERN has a successful experience with running Server Self Service Center (S3C) for virtual server provisioning which is based on Microsoft® Virtual Server 2005. With the introduction of Windows Server 2008 and its built-in hypervisor based virtualization (Hyper-V) there are new possibilities for the expansion of the current service. This paper describes the architecture of the redesigned virtual Server Self Service based on Hyper-V which provides dynamically scalable virtualized resources on demand as needed and outlines the possible implications on the future use of virtual machines at CERN.

  4. Variability and spectral variation of 3C 66A

    CERN Document Server

    Hu, Shao Ming; Guo, H Y; Zhou, X; Zhang, X; Zheng, Y G

    2013-01-01

    3C 66A was monitored by the BATC(Beijing-Arizona-Taipei-Connecticut) telescope from 2005 to 2008,1994 observations were obtained on 89 nights. Detailed research and analysis was performed on these observations in this paper. A long term burst occurred in the whole light curve. No intra-day variability was claimed in our campaign by intra-night light curve analysis. Time lag of shorter wavelenth preceding longer wavelength was shown by correlation analysis. The results showed that the optical spectral shape turned flatter when the source brightened, and the spectral variability indicator was bigger on shorter time-scale as determined by the color indices variation analysis.

  5. Photopolarimetry of Blazar 3C454.3 from MIRO

    Science.gov (United States)

    Baliyan, Ks; Ganesh, S.; Chandra, Sunil; Joshi, Uc

    2009-12-01

    The Blazar 3C 454.3 has been active in Gamma-rays, optical and X- rays since Sept. 2009 ( Atel #2181, #2200, #2201). Very recently, it has been reported to be flaring up in the optical, X-ray and gamma-ray energy regimes(ATel #2322; #2325; #2326; #2328; #2329; #2330; #2332). In Atel #2333, Sasada et al report optical behaviour of this source on Dec 1.9 with brightness (V=14.06+/-0.02 and degree of polarization 6.0+/-0.1% on the same epoch.

  6. The Infrared Detection of the Pulsar Wind Nebula in the Galactic Supernova Remnant 3C 58

    Science.gov (United States)

    Slane, P.; Helfand, D. J.; Reynolds, S. P.; Gaensler, B. M.; Lemiere, A.; Wang, Z.

    2008-03-01

    We present infrared observations of 3C 58 with the Spitzer Space Telescope and the Canada-France-Hawaii Telescope. Using the IRAC camera, we have imaged the entire source, which results in clear detections of the nebula at 3.6 and 4.5 μm. The derived flux values are consistent with extrapolation of the X-ray spectrum to the infrared band, demonstrating that any cooling break in the synchrotron spectrum must occur near the soft X-ray band. We also detect the torus surrounding PSR J0205+6449, the 65 ms pulsar that powers 3C 58. The torus spectrum requires a break between the infrared and X-ray bands, and perhaps multiple breaks. This complex spectrum, which is an imprint of the particles injected into the nebula, has considerable consequences for the evolution of the broadband spectrum of 3C 58. We illustrate these effects and discuss the impact of these observations on the modeling of broadband spectra of pulsar wind nebulae.

  7. The Infrared Detection of the Pulsar Wind Nebula in the Galactic Supernova Remnant 3C 58

    CERN Document Server

    Slane, P; Reynolds, S P; Gaensler, B M; Lemiere, A; Wang, Z

    2008-01-01

    We present infrared observations of 3C 58 with the Spitzer Space Telescope and the Canada-France-Hawaii Telescope. Using the IRAC camera, we have imaged the entire source resulting in clear detections of the nebula at 3.6 and 4.5 microns. The derived flux values are consistent with extrapolation of the X-ray spectrum to the infrared band, demonstrating that any cooling break in the synchrotron spectrum must occur near the soft X-ray band. We also detect the torus surrounding PSR J0205+6449, the 65 ms pulsar that powers 3C 58. The torus spectrum requires a break between the infrared and X-ray bands, and perhaps multiple breaks. This complex spectrum, which is an imprint of the particles injected into the nebula, has considerable consequences for the evolution of the broadband spectrum of 3C 58. We illustrate these effects and discuss the impact of these observations on the modeling of broadband spectra of pulsar wind nebulae.

  8. The Trails of Superluminal Jet Components in 3C111

    CERN Document Server

    Kadler, M; Perucho, M; Kovalev, Y Y; Homan, D C; Agudo, I; Kellermann, K I; Aller, M F; Aller, H D; Lister, M L; Zensus, J A

    2008-01-01

    In 1996, a major radio flux-density outburst occured in the broad-line radio galaxy 3C111. It was followed by a particularly bright plasma ejection associated with a superluminal jet component, which has shaped the parsec-scale structure of 3C111 for almost a decade. Here, we present results from 18 epochs of Very Long Baseline Array (VLBA) observations conducted since 1995 as part of the VLBA 2 cm Survey and MOJAVE monitoring programs. This major event allows us to study a variety of processes associated with outbursts of radio-loud AGN in much greater detail than has been possible in other cases: the primary perturbation gives rise to the formation of a leading and a following component, which are interpreted as a forward and a backward-shock. Both components evolve in characteristically different ways and allow us to draw conclusions about the work flow of jet-production events; the expansion, acceleration and recollimation of the ejected jet plasma in an environment with steep pressure and density gradien...

  9. Modelling the $\\gamma$-ray variability of 3C 273

    CERN Document Server

    Zheng, Y G; Huang, B R; Kang, S J

    2016-01-01

    We investigate MeV-GeV $\\gamma$-ray outbursts in 3C 273 in the frame of a time-dependent one-zone synchrotron self-Compton (SSC) model. In this model, electrons are accelerated to extra-relativistic energy through the stochastic particle acceleration and evolve with the time, nonthermal photons are produced by both synchrotron and inverse Compton scattering of synchrotron photons. Moreover, nonthermal photons during a quiescent are produced by the relativistic electrons in the steady state and those during a outburst are produced by the electrons whose injection rate is changed at some time interval. We apply the model to two exceptionally luminous $\\gamma$-ray outbursts observed by the Fermi-LAT from 3C 273 in September, 2009 and obtain the multi-wavelength spectra during the quiescent and during the outburst states, respectively. Our results show that the time-dependent properties of outbursts can be reproduced by adopting the appropriate injection rate function of the electron population.

  10. The high energy spectrum of 3C 273

    CERN Document Server

    Esposito, V; Jean, P; Tramacere, A; Türler, M; Lähteenmäki, A; Tornikoski, M

    2015-01-01

    Aims. The high energy spectrum of 3C 273 is usually understood in terms of inverse-Compton emission in a relativistic leptonic jet. This model predicts variability patterns and delays that could be tested with simultaneous observations from the radio to the GeV range. Methods. The instruments IBIS, SPI, JEM-X on board INTEGRAL, PCA on board RXTE, and LAT on board Fermi have enough sensitivity to follow the spectral variability of 3C 273 from the keV to the GeV. We looked for correlations between the different energy bands, including radio data at 37 GHz collected at the Mets\\"ahovi Radio Observatory and built quasi-simultaneous multiwavelength spectra in the high energy domain when the source is flaring either in the X-rays or in the {\\gamma} rays. Results. Both temporal and spectral analysis suggest a two-component model to explain the complete high energy spectrum. X-ray emission is likely dominated by a Seyfert-like component while the {\\gamma}-ray emission is dominated by a blazar-like component produced ...

  11. XMM-Newton observations of 3C 273

    CERN Document Server

    Page, K L; Done, C; O'Brien, P T; Reeves, J N; Sembay, S; Stuhlinger, M

    2004-01-01

    A series of nine XMM-Newton observations of the radio-loud quasar 3C 273 are presented, concentrating mainly on the soft excess. Although most of the individual observations do not show evidence for iron emission, co-adding them reveals a weak, broad line (EW ~ 56 eV). The soft excess component is found to vary, confirming previous work, and can be well fitted with multiple blackbody components, with temperatures ranging between ~40 and ~330 eV, together with a power-law. Alternatively, a Comptonisation model also provides a good fit, with a mean electron temperature of ~350 eV, although this value is higher when the soft excess is more luminous over the 0.5-10 keV energy band. In the RGS spectrum of 3C 273, a strong detection of the OVII He-alpha absorption line at zero redshift is made; this may originate in warm gas in the local intergalactic medium, consistent with the findings of both Fang et al. (2003) and Rasmussen et al. (2003).

  12. Photoluminescence Properties of Nanocrystalline 3C-SiC Films

    Institute of Scientific and Technical Information of China (English)

    YU Wei; LU Xue-qin; LU Wan-bing; HAN Li; FU Guang-sheng

    2006-01-01

    Nanocrystalline (nc) 3C-SiC films on the Si substrate were prepared by the helicon wave plasma enhanced chemical vapor deposition (HW-PECVD) technique. With the SiH4-CH4 gas flow ratio changing, the films exhibit different photoluminescence (PL) characteristics. Under the stoichiometric condition, the PL peak redshift from 470 nm to 515 nm is detected with the increase of excitation wavelength, which can be attributed to the quantum confinement effect radiation of 3C-SiC nanocrystals of different sizes. However, the appearance of an additional PL band at 436 nm in Si-rich film might be sourced back to the excess of Si defect centers in it. This is also the case for C-rich film for its PL band lying at 570 nm. The results above quoted indicate an important influence of gas flow ratio on the PL properties of the SiC films providing an effective guidance for analyzing the luminescence mechanism and exploring the high-efficiency light emission of the SiC films.

  13. The anatomy of a radio source hot spot : Very large baseline array imaging of 3C 205

    NARCIS (Netherlands)

    Lonsdale, CJ; Barthel, PD

    1998-01-01

    Total intensity and linear polarization Very Long Baseline Array (VLBA) images of the high-redshift quasar 3C 205 at a wavelength of 18 cm reveal a complex curved hot-spot structure with polarization percentages frequently as high as 70%. A one-sided jet is detected emerging from the central compone

  14. Crystal Structures of a Multidrug-Resistant Human Immunodeficiency Virus Type 1 Protease Reveal an Expanded Active-Site Cavity

    Energy Technology Data Exchange (ETDEWEB)

    Logsdon, Bradley C.; Vickrey, John F.; Martin, Philip; Proteasa, Gheorghe; Koepke, Jay I.; Terlecky, Stanley R.; Wawrzak, Zdzislaw; Winters, Mark A.; Merigan, Thomas C.; Kovari, Ladislau C. (Stanford); (WSU-MED); (NWU); (Stanford-MED)

    2010-03-08

    The goal of this study was to use X-ray crystallography to investigate the structural basis of resistance to human immunodeficiency virus type 1 (HIV-1) protease inhibitors. We overexpressed, purified, and crystallized a multidrug-resistant (MDR) HIV-1 protease enzyme derived from a patient failing on several protease inhibitor-containing regimens. This HIV-1 variant contained codon mutations at positions 10, 36, 46, 54, 63, 71, 82, 84, and 90 that confer drug resistance to protease inhibitors. The 1.8-{angstrom} crystal structure of this MDR patient isolate reveals an expanded active-site cavity. The active-site expansion includes position 82 and 84 mutations due to the alterations in the amino acid side chains from longer to shorter (e.g., V82A and I84V). The MDR isolate 769 protease 'flaps' stay open wider, and the difference in the flap tip distances in the MDR 769 variant is 12 {angstrom}. The MDR 769 protease crystal complexes with lopinavir and DMP450 reveal completely different binding modes. The network of interactions between the ligands and the MDR 769 protease is completely different from that seen with the wild-type protease-ligand complexes. The water molecule-forming hydrogen bonds bridging between the two flaps and either the substrate or the peptide-based inhibitor are lacking in the MDR 769 clinical isolate. The S1, S1', S3, and S3' pockets show expansion and conformational change. Surface plasmon resonance measurements with the MDR 769 protease indicate higher k{sub off} rates, resulting in a change of binding affinity. Surface plasmon resonance measurements provide k{sub on} and k{sub off} data (K{sub d} = k{sub off}/k{sub on}) to measure binding of the multidrug-resistant protease to various ligands. This MDR 769 protease represents a new antiviral target, presenting the possibility of designing novel inhibitors with activity against the open and expanded protease forms.

  15. 17 CFR 270.3c-3 - Definition of certain terms used in section 3(c)(1) of the Act with respect to certain debt...

    Science.gov (United States)

    2010-04-01

    ... investment companies. 270.3c-3 Section 270.3c-3 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.3c-3 Definition of certain... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Definition of certain...

  16. Milliarcsecond polarization structure of the superluminal quasar 3C 273

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, D.H.; Kollgaard, R.I.; Brown, L.F.; Gabuzda, D.C.; Wardle, J.F. C. (Brandeis Univ., Waltham, MA (USA))

    1990-09-01

    A 2 x 10 marcsec-resolution determination is presented for the total intensity and linear polarization structures of the superluminal quasar 3C 273 at 5 GHz. Substantial polarized flux was detected from several superluminal components of the jet, whose fractional polarization increased symmetrically with distance from the core; the most distant component is highly polarized and exhibits a highly ordered magnetic field. Within a few marcsec of the core, the inferred magnetic field orientation varies rapidly with position along the jet. The primarily longitudinal magnetic field orientation is concluded to become established within 20 marcsec of the core. A highly disorganized magnetic field is the most plausible explanation for the low degree of polarization in the innermost regions of the jet. 48 refs.

  17. Milliarcsecond polarization structure of the superluminal quasar 3C 273

    International Nuclear Information System (INIS)

    A 2 x 10 marcsec-resolution determination is presented for the total intensity and linear polarization structures of the superluminal quasar 3C 273 at 5 GHz. Substantial polarized flux was detected from several superluminal components of the jet, whose fractional polarization increased symmetrically with distance from the core; the most distant component is highly polarized and exhibits a highly ordered magnetic field. Within a few marcsec of the core, the inferred magnetic field orientation varies rapidly with position along the jet. The primarily longitudinal magnetic field orientation is concluded to become established within 20 marcsec of the core. A highly disorganized magnetic field is the most plausible explanation for the low degree of polarization in the innermost regions of the jet. 48 refs

  18. Radio Spectral Index and Expansion of 3C58

    CERN Document Server

    Bietenholz, M F; Weiler, K W

    2001-01-01

    We present new observations of the plerionic supernova remnant 3C58 with the VLA at 74 and 327 MHz. In addition, we re-reduced earlier observations at 1.4 and 4.9 GHz taken in 1973 and 1984. Comparing these various images, we find that: 1. the remnant has a flat and relatively uniform spectral index distribution, 2. any expansion of the remnant with time is significantly less than that expected for uniform, undecelerated expansion since the generally accepted explosion date in 1181 A.D., and 3. there is no evidence for a non-thermal synchrotron emission shell generated by a supernova shock wave, with any such emission having a surface brightness of <1 x 10^(-21) W / (m^2 Hz sr) at 327 MHz.

  19. Structural variability of 3C 111 on parsec scales

    CERN Document Server

    Großberger, C; Wilms, J; Müller, C; Beuchert, T; Ros, E; Ojha, R; Aller, M; Aller, H; Angelakis, E; Fuhrmann, L; Nestoras, I; Schmidt, R; Zensus, J A; Krichbaum, T P; Ungerechts, H; Sievers, A; Riquelme, D

    2011-01-01

    We discuss the parsec-scale structural variability of the extragalactic jet 3C 111 related to a major radio flux density outburst in 2007. The data analyzed were taken within the scope of the MOJAVE, UMRAO, and F-GAMMA programs, which monitor a large sample of the radio brightest compact extragalactic jets with the VLBA, the University of Michigan 26 m, the Effelsberg 100 m, and the IRAM 30 m radio telescopes. The analysis of the VLBA data is performed by fitting Gaussian model components in the visibility domain. We associate the ejection of bright features in the radio jet with a major flux-density outburst in 2007. The evolution of these features suggests the formation of a leading component and multiple trailing components.

  20. Synthesis of the C3-C12 Fragment of Laulimalide

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Hyo Won; Hong, Joong Yeoun [Chungbuk National University, Cheongju (Korea, Republic of)

    2003-11-15

    We have reported the successful synthesis of the C3-C12 fragment of laulimalide using the introduction of a dihydropyran ring after the elongation of a side chain. Our on-going synthetic effort toward the synthesis of laulimalide will be reported in the near future. The antimiototic 20-membered macrolide laulimalide also known as fijanolide B, isolated from sea sponges such as Cacospongia mycofijiensis, Hyatella sp. Spongia mycofijiensis, and Fasciospongia rimosa, shows a potent biological activity against cell lines resistant to paclitaxel or epothilones since laulimalide binds to a site of microtubules in a mode distinct from taxoids. The stabilization of polymerization by laulimalide leads to the inhibition of miototic spindle and apoptosis of cells.

  1. Immobilization of bacterial proteases on water-solved polymer by means of electron beam

    International Nuclear Information System (INIS)

    Possibility of electron beam usage for proteases' immobilization on 1,4-polyalkylene oxide (1,4-PAO) was studied to obtain biologically active complex for multi-purpose usage. It is shown that immobilization of Bacillus Subtilis protease takes place due to free-radical linking of enzyme and carrier with formation of mycellium-like structures. Immobilization improves heat resistance of enzyme up to 60oC without substrate and up to 80oC in presence of substrate, widens range of pH activity in comparison with non-immobilized forms. Immobilized proteases do not contain peroxides or long-live radicals. Our results permitted to create technologies for production of medical and veterinary preparations, active components for wool washing agents and leather fabrication technology. (Author)

  2. Structural Evidence for Regulation and Specificity of Flaviviral Proteases and Evolution of the Flaviviridae Fold

    Energy Technology Data Exchange (ETDEWEB)

    Aleshin,A.; Shiryaev, S.; Strongin, A.; Liddington, R.

    2007-01-01

    Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.

  3. NS3 Protease from Hepatitis C Virus: Biophysical Studies on an Intrinsically Disordered Protein Domain

    Directory of Open Access Journals (Sweden)

    Adrian Velazquez-Campoy

    2013-06-01

    Full Text Available The nonstructural protein 3 (NS3 from the hepatitis C virus (HCV is responsible for processing the non-structural region of the viral precursor polyprotein in infected hepatic cells. NS3 protease activity, located at the N-terminal domain, is a zinc-dependent serine protease. A zinc ion, required for the hydrolytic activity, has been considered as a structural metal ion essential for the structural integrity of the protein. In addition, NS3 interacts with another cofactor, NS4A, an accessory viral protein that induces a conformational change enhancing the hydrolytic activity. Biophysical studies on the isolated protease domain, whose behavior is similar to that of the full-length protein (e.g., catalytic activity, allosteric mechanism and susceptibility to inhibitors, suggest that a considerable global conformational change in the protein is coupled to zinc binding. Zinc binding to NS3 protease can be considered as a folding event, an extreme case of induced-fit binding. Therefore, NS3 protease is an intrinsically (partially disordered protein with a complex conformational landscape due to its inherent plasticity and to the interaction with its different effectors. Here we summarize the results from a detailed biophysical characterization of this enzyme and present new experimental data.

  4. SCREENING OF PROTEASE ENZYME BY CONSTRUCTION OF METAGENOMIC LIBRARY FROM MARINE SOIL SEDIMENTS

    Directory of Open Access Journals (Sweden)

    R.PRABAVATHI

    2012-07-01

    Full Text Available Nonetheless, the cultivable microorganisms constituting these resources correspond to only a small fraction of the microbial diversity less than 1% of the microorganisms in various environments are readily cultivable (Amann et al., 1995. This limits the range of a search for new biocatalysts for the bioprocess industry, so the use of complex communities and the effort to overcome the problem of noncultivability attract not only scientific attention but also biotechnological innovation. Methods have been developed and used toovercome the non-cultivability of environmental microorganisms for biotechnology, namely cloning and the expression of metagenomes in suitable expression hosts. Proteases are present in all living forms as they are involved in various metabolic processes. They are mainly involved in hydrolysis of the peptide bonds (Gupta et al., 2002. Proteases find a wide range of applications in food, pharmaceutical, leather and textile, detergent, diagnostics industries and also in waste management. In order to discover new proteases from metagenomiclibraries, we screened for proteolytic activity from a constructed metagenomic library by direct cloning of environmental DNA of large DNA inserts. A novel gene encoding proteolytic enzyme was picked up,sequenced, expressed in E. coli and characterized. Several microbial proteases from the culturable organisms have been characterized. However, very few proteases have been identified through culture independent metagenomic approach.

  5. Anisotropies in the HI gas distribution toward 3C 196

    Science.gov (United States)

    Kalberla, P. M. W.; Kerp, J.

    2016-10-01

    Context. The local Galactic Hi gas was found to contain cold neutral medium (CNM) filaments that are aligned with polarized dust emission. These filaments appear to be dominated by the magnetic field and in this case turbulence is expected to show distinct anisotropies. Aims: We use the Galactic Effelsberg-Bonn Hi Survey (EBHIS) to derive 2D turbulence spectra for the Hi distribution in direction to 3C 196 and two more comparison fields. Methods: Prior to Fourier transform we apply a rotational symmetric 50% Tukey window to apodize the data. We derive average as well as position angle dependent power spectra. Anisotropies in the power distribution are defined as the ratio of the spectral power in orthogonal directions. Results: We find strong anisotropies. For a narrow range in position angle, in direction perpendicular to the filaments and the magnetic field, the spectral power is on average more than an order of magnitude larger than parallel. In the most extreme case the anisotropy reaches locally a factor of 130. Anisotropies increase on average with spatial frequency as predicted by Goldreich & Sridhar (1995, ApJ, 438, 763), at the same time the Kolmogorov spectral index remains almost unchanged. The strongest anisotropies are observable for a narrow range in velocity and decay with a power law index close to -8/3, almost identical to the average isotropic spectral index of -2.9 Hi filaments, associated with linear polarization structures in LOFAR observations in direction to 3C 196, show turbulence spectra with marked anisotropies. Decaying anisotropies appear to indicate that we witness an ongoing shock passing the Hi and affecting the observed Faraday depth.

  6. Cleavage entropy as quantitative measure of protease specificity.

    Directory of Open Access Journals (Sweden)

    Julian E Fuchs

    2013-04-01

    Full Text Available A purely information theory-guided approach to quantitatively characterize protease specificity is established. We calculate an entropy value for each protease subpocket based on sequences of cleaved substrates extracted from the MEROPS database. We compare our results with known subpocket specificity profiles for individual proteases and protease groups (e.g. serine proteases, metallo proteases and reflect them quantitatively. Summation of subpocket-wise cleavage entropy contributions yields a measure for overall protease substrate specificity. This total cleavage entropy allows ranking of different proteases with respect to their specificity, separating unspecific digestive enzymes showing high total cleavage entropy from specific proteases involved in signaling cascades. The development of a quantitative cleavage entropy score allows an unbiased comparison of subpocket-wise and overall protease specificity. Thus, it enables assessment of relative importance of physicochemical and structural descriptors in protease recognition. We present an exemplary application of cleavage entropy in tracing substrate specificity in protease evolution. This highlights the wide range of substrate promiscuity within homologue proteases and hence the heavy impact of a limited number of mutations on individual substrate specificity.

  7. Dataset of cocoa aspartic protease cleavage sites.

    Science.gov (United States)

    Janek, Katharina; Niewienda, Agathe; Wöstemeyer, Johannes; Voigt, Jürgen

    2016-09-01

    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. PMID:27508221

  8. 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.

  9. Structure-Guided Design and Optimization of Dipeptidyl Inhibitors of Norovirus 3CL Protease. Structure-Activity Relationships and Biochemical, X-ray Crystallographic, Cell-Based, and In Vivo Studies

    Science.gov (United States)

    Kankanamalage, Anushka C. Galasiti; Kim, Yunjeong; Weerawarna, Pathum M.; Uy, Roxanne Adeline Z.; Damalanka, Vishnu C.; Mandadapu, Sivakoteswara Rao; Alliston, Kevin R.; Mehzabeen, Nurjahan; Battaile, Kevin P.; Lovell, Scott; Chang, Kyeong-Ok; Groutas, William C.

    2015-01-01

    Norovirus infection constitutes the primary cause of acute viral gastroenteritis. There are currently no vaccines or norovirus-specific antiviral therapeutics available for the management of norovirus infection. Norovirus 3C-like protease is essential for viral replication, consequently, inhibition of this enzyme is a fruitful avenue of investigation that may lead to the emergence of anti-norovirus therapeutics. We describe herein the optimization of dipeptidyl inhibitors of norovirus 3C-like protease using iterative SAR, X-ray crystallographic, and enzyme and cell-based studies. We also demonstrate herein in vivo efficacy of an inhibitor using the murine model of norovirus infection. PMID:25761614

  10. Hydrolysis of Fish Protein by Analkaline Protease

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Cod muscle protein was hydrolyzed by an alkaline protease in our study. The influences of hydrolysis temperature,fish protein concentration,and ratio of protease addition to protein amount on its degree of hy drolysis (DH) of protein were studied in details by applying dual quadratic rotary combinational design. The final results showed that more than 84% cod muscle protein could be hydrolyzed and recovered. Cod protein hydrolysate thus obtained had a balanced amino acid composition and mainly consisted of small peptides with molecule weight less than 6900 dalton.

  11. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

    This thesis describes the design and synthesis of peptide-based serine protease inhibitors. The targeted protease, urokinase-type plasminogen activator (uPA) activates plasminogen, which plays a major role in cancer metastasis. The peptide upain-2 (S 1 ,S 12-cyclo-AcCSWRGLENHAAC-NH2) is a highly...... increased. Finally, the effect of multivalent display of upain-2 was investigated. Several dimers of upain-2 were made and the attachment of upain-2 via the Copper-catalyzed Azide-Alkyne Cycloaddition (CuAAC) onto an alkyne functionalized carbohydrate scaffold was investigated. Besides the synthesis...

  12. 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...... of this peptidic inhibitor, a concept different from conventional attempts at improving inhibitor affinity by reducing the entropic burden....

  13. Serine Proteases an Ab Initio Molecular Dynamics Study

    CERN Document Server

    De Santis, L

    1999-01-01

    In serine proteases (SP's), the H-bond between His-57 and Asp-102, and that between Gly-193 and the transition state intermediate play a crucial role for enzymatic function. To shed light on the nature of these interactions, we have carried out ab initio molecular dynamics simulations on complexes representing adducts between the reaction intermediate and elastase (one protein belonging to the SP family). Our calculations indicate the presence of a low--barrier H-bond between His-57 and Asp-102, in complete agreement with NMR experiments on enzyme--transition state analog complexes. Comparison with an ab initio molecular dynamics simulation on a model of the substrate--enzyme adduct indicates that the Gly-193--induced strong stabilization of the intermediate is accomplished by charge/dipole interactions and not by H-bonding as previously suggested. Inclusion of the protein electric field in the calculations does not affect significantly the charge distribution.

  14. 50 CFR Table 3c to Part 680 - Crab Product Codes for Economic Data Reports

    Science.gov (United States)

    2010-10-01

    ... EXCLUSIVE ECONOMIC ZONE OFF ALASKA Pt. 680, Table 3c Table 3c to Part 680—Crab Product Codes for Economic... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Crab Product Codes for Economic Data Reports 3c Table 3c to Part 680 Wildlife and Fisheries FISHERY CONSERVATION AND MANAGEMENT,...

  15. 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....

  16. Detection of protease and protease activity using a single nanoscrescent SERS probe

    Science.gov (United States)

    Liu, Gang L.; Ellman, Jonathan A.; Lee, Luke P.; Chen, Fanqing Frank

    2013-01-29

    This invention pertains to the in vitro detection of proteases using a single peptide-conjugate nanocrescent surface enhanced Raman scattering (SERS) probes with at least nanomolar sensitivity. The probe enables detection of proteolytic activity in extremely small volume and at low concentration. In certain embodiments the probes comprise an indicator for the detection of an active protease, where the indicator comprises a nanocrescent attached to a peptide, where said peptide comprises a recognition site for the protease and a Raman tag attached to the peptide.

  17. Correlated variability in the blazar 3C 454.3

    CERN Document Server

    Bonning, E W; Urry, C M; Buxton, M; Fossati, G; Maraschi, L; Coppi, P; Scalzo, R; Isler, J; Kaptur, A

    2008-01-01

    The blazar 3C 454.3 was revealed by the Fermi Gamma-ray Space Telescope to be in an exceptionally high flux state in July 2008. Accordingly, we performed a multi-wavelength monitoring campaign on this blazar using IR and optical observations from the SMARTS telescopes, optical, UV and X-ray data from the Swift satellite, and public-release gamma-ray data from Fermi. We find an excellent correlation between the IR, optical, UV and gamma-ray light curves, with a time lag of less than one day. The amplitude of the infrared variability is comparable to that in gamma-rays, and larger than at optical or UV wavelengths. The X-ray flux is not strongly correlated with either the gamma-rays or longer wavelength data. These variability characteristics find a natural explanation in the external Compton model, in which electrons with Lorentz factor gamma~10^(3-4) radiate synchrotron emission in the infrared-optical and also scatter accretion disk or emission line photons to gamma-ray energies, while much cooler electrons ...

  18. The dynamics of the giant radio galaxy 3C 457

    CERN Document Server

    Konar, C; Croston, J H; Saikia, D J

    2009-01-01

    We present multi-frequency radio observations with the Giant Metrewave Radio Telescope and Very Large Array, and X-ray observations with the X-ray Multi-Mirror Mission ({\\it XMM-Newton}) telescope of the giant radio source (GRS) 3C 457. We have detected the core, lobes and the environment of the GRS in X-ray. We examine the relationships between the radio and X-ray emission, determine the radio spectrum over a large frequency range and attribute the X-ray emission from the lobes to the inverse-Compton scattering of cosmic microwave background (CMB) photons. The magnetic field strength of the lobes is very close to the equipartition value. Both the lobes are in pressure balance near the hotspots and apparently under-pressured towards the core. The X-ray spectrum of the core of the GRS consists of an unabsorbed soft power-law component and a heavily absorbed hard power-law component. The soft unabsorbed component is likely to be related to the radio jets. There is no strong evidence of Fe K$\\alpha$ emission lin...

  19. The blue-bump of 3C 273

    CERN Document Server

    Paltani, S; Walter, R

    1998-01-01

    We present optical and ultraviolet observations of 3C273 covering the whole life of the IUE satellite. We analyze the variability properties of the light curves, and find that two variable components, written B and R respectively, must contribute to the blue-bump emission in this object. The B component produces most of the variability in the ultraviolet domain. A maximum time scale of variability of about 2 yr identical at all wavelengths is found. If discrete events produce this component, the event rate is ~3-30 yr^-1. Assuming an isotropic emission, each event must liberate about 10^51 erg in the form of optical-to-ultraviolet radiation. The spectral properties of the B component suggest that reprocessing on a truncated disk, or partially- thick bremsstrahlung may be the emission mechanism. We find evidence of a lag of a few days between the light curves of the B component at optical and ultraviolet wavelengths. Neither the variability properties, nor the spectral properties of the R component can be accu...

  20. Suzaku observation of the giant radio galaxy 3C 326

    CERN Document Server

    Isobe, Naoki; Gandhi, Poshak; Hayato, Asami; Nagai, Hiroshi; Hada, Kazuhiro; Seta, Hiromi; Matsuta, Keiko

    2009-01-01

    A Suzaku observation of a giant radio galaxy, 3C 326, which has a physical size of about 2 Mpc, was conducted on 2008 January 19 -- 21. In addition to several X-ray sources, diffuse emission was significantly detected associated with its west lobe, but the east lobe was contaminated by an unidentified X-ray source WARP J1552.4+2007. After careful evaluation of the X-ray and Non X-ray background, the 0.4 -- 7 keV X-ray spectrum of the west lobe is described by a power-law model. The photon index and 1 keV flux density was derived as $1.82_{-0.24}^{+0.26}\\pm0.04$ and $19.4_{-3.2}^{+3.3}\\pm 3.0$ nJy, respectively, where the first and second errors represent the statistical and systematic ones. The diffuse X-rays were attributed to be inverse Compton radiation by the synchrotron radio electrons scattering off the cosmic microwave background photons. This radio galaxy is the largest among those with lobes detected through inverse Compton X-ray emission. A comparison of the radio to X-ray fluxes yields the energy d...

  1. Spectroscopic monitoring of the Blazar 3C 454.3

    CERN Document Server

    Benítez, E; Raiteri, C M; Villata, M; Dultzin, D; Martínez, O; Perez-Camargo, B; Torrealba, J

    2009-01-01

    We performed an optical spectroscopic monitoring of the blazar 3C 454.3 from September 2003 to July 2008. Sixteen optical spectra were obtained during different runs, which constitute the first spectroscopic monitoring done in the rest-frame UV region (z=0.859). An overall flux variation of the MgII (2800 A) by a factor ~ 3 was observed, while the corresponding UV continuum (F_cont at 3000 A) changed by a factor ~ 14. The MgII emission lines respond proportionally to the continuum variations when the source is in a low-activity state. In contrast, near the optical outbursts detected in 2005 and 2007, the MgII emission lines showed little response to the continuum flux variations. During the monitored period the UV FeII flux changed by a factor ~ 6 and correlated with F_cont (r = 0.92). A negative correlation between EW(Mg II) and F_cont was found, i.e. the so-called "Intrinsic Baldwin Effect".

  2. The Extended Line Region of 3C 299

    CERN Document Server

    Feinstein, C; Martel, A R; Sparks, W B; McCarthy, P J; Feinstein, Carlos; Martel, Andre R.; Sparks, William B.; Carthy, Patrick J. Mc

    1999-01-01

    We present results of HST observations of the radio galaxy 3C 299. The broad-band F702W (R) and F555W (V) images (WFPC2/PC) show an elliptical galaxy, with a comet-like structure extending to the NE in the radio jet direction. The [OIII]$\\lambda$5007 emission line map, shows a bi-conical structure centered on the nucleus, that overlaps the structure found in the broad-band filters. The radio core coincides with the center of the bi-conical structure and the radio axes are aligned with the direction of the cones. These data show clear evidence of a strong interaction between the radio jet and the NE morphology of the galaxy. We show evidence that this NE region is an ENLR; the line-ratio diagnostics show that models involving gas shocked by the radio-jet plus ionization from a precursor HII region, produced itself by the ionizing photons of the postshocked gas on the preshocked gas provide a good match to the observations. We investigate the spatial behavior of the ionizing parameter $U$, by determining the [O...

  3. Organic functionalization of 3C-SiC surfaces.

    Science.gov (United States)

    Schoell, Sebastian J; Sachsenhauser, Matthias; Oliveros, Alexandra; Howgate, John; Stutzmann, Martin; Brandt, Martin S; Frewin, Christopher L; Saddow, Stephen E; Sharp, Ian D

    2013-02-01

    We demonstrate the functionalization of n-type (100) and (111) 3C-SiC surfaces with organosilanes. Self-assembled monolayers (SAMs) of amino-propyldiethoxymethylsilane (APDEMS) and octadecyltrimethoxysilane (ODTMS) are formed via wet chemical processing techniques. Their structural, chemical, and electrical properties are investigated using static water contact angle measurements, atomic force microscopy, and X-ray photoelectron spectroscopy, revealing that the organic layers are smooth and densely packed. Furthermore, combined contact potential difference and surface photovoltage measurements demonstrate that the heterostructure functionality and surface potential can be tuned by utilizing different organosilane precursor molecules. Molecular dipoles are observed to significantly affect the work functions of the modified surfaces. Furthermore, the magnitude of the surface band bending is reduced following reaction of the hydroxylated surfaces with organosilanes, indicating that partial passivation of electrically active surface states is achieved. Micropatterning of organic layers is demonstrated by lithographically defined oxidation of organosilane-derived monolayers in an oxygen plasma, followed by visualization of resulting changes of the local wettability, as well as fluorescence microscopy following immobilization of fluorescently labeled BSA protein. PMID:23357505

  4. Anisotropies in the HI gas distribution toward 3C196

    CERN Document Server

    Kalberla, P M W

    2016-01-01

    The local Galactic HI gas was found to contain cold neutral medium (CNM) filaments that are aligned with polarized dust emission. These filaments appear to be dominated by the magnetic field and in this case turbulence is expected to show distinct anisotropies. We use the Galactic Effelsberg--Bonn HI Survey (EBHIS) to derive 2D turbulence spectra for the HI distribution in direction to 3C196 and two more comparison fields. Prior to Fourier transform we apply a rotational symmetric 50% Tukey window to apodize the data. We derive average as well as position angle dependent power spectra. Anisotropies in the power distribution are defined as the ratio of the spectral power in orthogonal directions. We find strong anisotropies. For a narrow range in position angle, in direction perpendicular to the filaments and the magnetic field, the spectral power is on average more than an order of magnitude larger than parallel. In the most extreme case the anisotropy reaches locally a factor of 130. Anisotropies increase on...

  5. 3C 273 with NuSTAR: Unveiling the AGN

    CERN Document Server

    Madsen, Kristin K; Walton, Dominic J; Harrison, Fiona A; Ballantyne, David R; Boggs, Steve E; Brenneman, Laura W; Christensen, Finn E; Craig, William W; Fabian, Andrew C; Forster, Karl; Grefenstette, Brian W; Guainazzi, Matteo; Hailey, Charles J; Madejski, Greg M; Matt, Giorgio; Stern, Daniel; Zhang, William W

    2015-01-01

    We present results from a 244 ks $NuSTAR$ observation of 3C 273, obtained during a cross-calibration campaign with the $Chandra$, $INTEGRAL$, $Suzaku$, $Swift$, and $XMM-Newton$ observatories. We show that the spectrum, when fit with a power-law model using data from all observatories except $INTEGRAL$ over the 1-78 keV band, leaves significant residuals in the $NuSTAR$ data between 30-78 keV. The $NuSTAR$ 3-78 keV spectrum is well-described by an exponentially cutoff power-law ($\\Gamma = 1.646 \\pm 0.006$, E$_\\mathrm{cutoff} = 202_{-34}^{+51}$ keV) with a weak reflection component from cold, dense material. There is also evidence for a weak ($EW = 23 \\pm 11$ eV) neutral iron line. We interpret these features as arising from coronal emission plus reflection off an accretion disk or distant material. Beyond 80 keV $INTEGRAL$ data show clear excess flux relative to an extrapolation of the AGN model fit to $NuSTAR$. This high-energy power-law is consistent with the presence of a beamed jet, which begins to domina...

  6. The effect of high glucose levels on the hypermethylation of protein phosphatase 1 regulatory subunit 3C (PPP1R3C) gene in colorectal cancer

    Indian Academy of Sciences (India)

    Soo Kyung Lee; Ji Wook Moon; Yong Woo Lee; Jung Ok Lee; Su Jin Kim; Nami Kim; Jin Kim; Hyeon Soo Kim; Sun-Hwa Park

    2015-03-01

    DNA methylation is an epigenetic event that occurs frequently in colorectal cancer (CRC). Increased glucose level is a strong risk factor for CRC. Protein phosphatase 1 regulatory subunit 3C (PPP1R3C) modulates glycogen metabolism, particularly glycogen synthesis. The aim of this study was to investigate the effect of high glucose levels on DNA methylation of PPP1R3C in CRC. PPP1R3C was significantly hypermethylated in CRC tissues (76/105, 72.38%, < 0.05) and colon cancer cell lines ( < 0.05). CRC tissues obtained from patients with high glucose levels showed that the methylation of PPP1R3C was lower than in patients who had normal levels of glucose. When DLD-1 cells were cultured under conditions of high glucose, the methylation of PPP1R3C was repressed. The expression of PPP1R3C was inversely related to methylation status. In addition, a promoter luciferase assay showed that the transcriptional activity of PPP1R3C was increased in high glucose culture conditions. The number of cells decreased when PPP1R3C was silenced in DLD-1 cells. These results suggest that PPP1R3C, a novel hypermethylated gene in CRC, may play a critical role in cancer cell growth in association with glucose levels.

  7. Overexpression of Elastin Affects the Protease to Anti-Protease Balance and Protects Mice from Colitis. : Elafin protects from colitis

    OpenAIRE

    Motta*, Jean-Paul; Magne, Laurent; Descamps, Delphyne; Rolland, Corinne; Squarzoni-Dale, Camila; Rousset, Perrine; Balloy, Viviane; Huerre, Michel; Jenne, Dieter; Wartelle, Julien; Belaaouaj, Azzaq; Mas, Emmanuel; Vinel, Jean-Pierre; Alric, Laurent; Chignard, Michel

    2010-01-01

    BACKGROUND & AIMS:: Colon tissues of patients with inflammatory bowel disease (IBD) have been reported to have increased proteolytic activity, but no studies have clearly addressed the protease to anti-protease balance in the pathogenesis of colitis. We investigated the role of Elafin, a serine protease inhibitor expressed by skin and mucosal surfaces in human inflammatory conditions, and the proteases neutrophil elastase (NE) and proteinase-3 (PR-3), in mice with colitis. METHODS:: We studie...

  8. MICROSPHERE-BASED FLOW CYTOMETRY PROTEASE ASSAYS FOR USE IN PROTEASE ACTIVITY DETECTION AND HIGH-THROUGHPUT SCREENING

    OpenAIRE

    Saunders, Matthew J.; Edwards, Bruce S.; Zhu, Jingshu; Sklar, Larry A.; Graves, Steven W.

    2010-01-01

    This protocol describes microsphere-based protease assays for use in flow cytometry and high-throughput screening. This platform measures a loss of fluorescence from the surface of a microsphere due to the cleavage of an attached fluorescent protease substrate by a suitable protease enzyme. The assay format can be adapted to any site or protein specific protease of interest and results can be measured in both real time and as end point fluorescence assays on a flow cytometer. End point assays...

  9. Bacterial proteases: targets for diagnostics and therapy

    NARCIS (Netherlands)

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

    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 po

  10. Characterization of a secreted Chlamydia protease

    DEFF Research Database (Denmark)

    Shaw, Allan C; Vandahl, Brian; Larsen, Martin Røssel;

    2002-01-01

    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...... fragments of CPAF exist in C. pneumoniae as well as in C. trachomatis....

  11. 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.

  12. Site-1 protease is required for cartilage development in zebrafish.

    Science.gov (United States)

    Schlombs, Kornelia; Wagner, Thomas; Scheel, Jochen

    2003-11-25

    gonzo (goz) is a zebrafish mutant with defects in cartilage formation. The goz phenotype comprises cartilage matrix defects and irregular chondrocyte morphology. Expression of endoderm, mesoderm, and cartilage marker genes is, however, normal, indicating a defect in chondrocyte morphogenesis. The mutated gene responsible for the goz phenotype, identified by positional cloning and confirmed by phosphomorpholino knockdown, encodes zebrafish site-1 protease (s1p). S1P has been shown to process and activate sterol regulatory element-binding proteins (SREBPs), which regulate expression of key enzymes of lipid biosynthesis or transport. This finding is consistent with the abnormal distribution of lipids in goz embryos. Knockdown of site-2 protease, which is also involved in activation of SREBPs, results in similar lipid and cartilage phenotypes as S1P knockdown. However, knockdown of SREBP cleavage-activating protein, which forms a complex with SREBP and is essential for S1P cleavage, results only in lipid phenotypes, whereas cartilage appears normal. This indicates that the cartilage phenoptypes of goz are caused independently of the lipid defects. PMID:14612568

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

    OpenAIRE

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

    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 profi...

  14. Multiwavelength Observations of 3C66A in 2003

    Science.gov (United States)

    Boettcher, M.; Joshi, M.; Fossati, G.; Smith, I. A.; Mukherjee, R.; Bramel, D.; Cui, W.; WEBT Collaboration

    2004-08-01

    The radio-selected BL Lac object 3C66A was the target of an intensive multiwavelength observing campaign in the last quarter of 2003 and early 2004. It was monitored by the Whole Earth Blazar Telescope (WEBT) collaboration of optical observers, in tandem with 20 X-ray monitoring observations by the Rossi X-Ray Timing Explorer (RXTE), VHE gamma-ray observations by STACEE and VERITAS, and long-term monitoring at radio frequencies. In addition, 9 high-spatial-resolution observations using the VLA are being carried out during the campaign and throughout the year 2004 to follow possible structural changes of the source. A gradual brightening of the source over the course of the campaign was observed at all optical frequencies, culminating in a very bright flare at the end of January 2004. Optical light curves indicate intraday microvariability on time scales down to about 1.3 hours. No significant color-magnitude correlation for the entire data set was evident, but there is a slight indication of a hardness - intensity anti-correlation on intraday time scales. The X-ray spectrum is consistent with a power-law with a photon spectral index of ˜ 2.1, indicating that the RXTE energy band might be located right at the intersection of the synchrotron and the high-energy emission components of the broadband spectral energy distribution. No significant flux or spectral variability at X-ray energies was detected. We extracted snapshot spectral energy distributions at various times throughout the campaign, and present first spectral fits to those SEDs. This work was partially supported by NASA RXTE GO grant no. NNG 04GB13G.

  15. Crystal Structures of the Histo-Aspartic Protease (HAP) from Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bhaumik, Prasenjit; Xiao, Huogen; Parr, Charity L.; Kiso, Yoshiaki; Gustchina, Alla; Yada, Rickey Y.; Wlodawer, Alexander; (Guelph); (Kyoto); (NCI)

    2009-08-07

    The structures of recombinant histo-aspartic protease (HAP) from malaria-causing parasite Plasmodium falciparum as apoenzyme and in complex with two inhibitors, pepstatin A and KNI-10006, were solved at 2.5-, 3.3-, and 3.05-{angstrom} resolutions, respectively. In the apoenzyme crystals, HAP forms a tight dimer not seen previously in any aspartic protease. The interactions between the monomers affect the conformation of two flexible loops, the functionally important 'flap' (residues 70-83) and its structural equivalent in the C-terminal domain (residues 238-245), as well as the orientation of helix 225-235. The flap is found in an open conformation in the apoenzyme. Unexpectedly, the active site of the apoenzyme contains a zinc ion tightly bound to His32 and Asp215 from one monomer and to Glu278A from the other monomer, with the coordination of Zn resembling that seen in metalloproteases. The flap is closed in the structure of the pepstatin A complex, whereas it is open in the complex with KNI-10006. Although the binding mode of pepstatin A is significantly different from that in other pepsin-like aspartic proteases, its location in the active site makes unlikely the previously proposed hypothesis that HAP is a serine protease. The binding mode of KNI-10006 is unusual compared with the binding of other inhibitors from the KNI series to aspartic proteases. The novel features of the HAP active site could facilitate design of specific inhibitors used in the development of antimalarial drugs.

  16. Investigating the X-ray and Gamma-ray Properties of the Galactic Supernova Remnants Kes 69, 3C 396, 3C 400.2

    CERN Document Server

    Ergin, Tülün; Yamazaki, Ryo

    2016-01-01

    Kes 69, 3C 396, and 3C 400.2 are mixed-morphology (MM) Galactic supernova remnants (SNRs), where Kes 69 and 3C 396 are interacting with molecular clouds (MCs). Previous X-ray studies showed that the emission from these SNRs is thermal. It has been suggested that MM SNRs interacting with MCs are potential candidates for recombining plasma (RP) in X-rays and hadronic gamma-ray emission. Recently, Chandra observations revealed signs of RP in 3C 400.2. Our preliminary analyses show that the X-ray emission of NW and SE region of 3C 400.2 arises from recombining plasma. We detected GeV gamma-ray emission from Kes 69 and 3C 396 above 5$\\sigma$.

  17. Serine Protease Autotransporters of Enterobacteriaceae (SPATEs: Biogenesis and Function

    Directory of Open Access Journals (Sweden)

    Nathalie Dautin

    2010-05-01

    Full Text Available Serine Protease Autotransporters of Enterobacteriaceae (SPATEs constitute a large family of proteases secreted by Escherichia coli and Shigella. SPATEs exhibit two distinct proteolytic activities. First, a C-terminal catalytic site triggers an intra-molecular cleavage that releases the N-terminal portion of these proteins in the extracellular medium. Second, the secreted N-terminal domains of SPATEs are themselves proteases; each contains a canonical serine-protease catalytic site. Some of these secreted proteases are toxins, eliciting various effects on mammalian cells. Here, we discuss the biogenesis of SPATEs and their function as toxins.

  18. Bacterial retropepsin-like proteases : the evidence from Legionella pneumophila

    OpenAIRE

    Teixeira, Paulo Alexandre Gonçalves

    2013-01-01

    A familia A2 de proteases aspárticas é constituida maioritariamente por proteases encontradas em retrovírus – as retropepsinas. As teorias evolutivas inerentes a estas proteases normalmente referem que estarão relacionadas com proteases do tipo pepsina pertencentes à familia A1 de proteases aspárticas. Pela primeira teoria (geralmente a mais aceite), durante a infeção de uma célula eucariota por um retrovírus, o gene da retropepsina terá sofrido duplicação e fusão dando orig...

  19. The Expression of Soluble and Active Recombinant Haemophilus influenzae IgA1 Protease in E. coli

    Directory of Open Access Journals (Sweden)

    Shinong Long

    2010-01-01

    Full Text Available Immunoglobulin A1 (IgA1 proteases from Haemophilus influenzae are extracellular proteases that specifically cleave the hinge region of human IgA1, the predominant class of immunoglobulin present on mucosal membranes. The IgA1 proteases may have the potential to cleave IgA1 complexes in the kidney and be a therapeutic agent for IgA1 nephropathy (IgAN, a disease characterized by deposition of the IgA1 antibody in the glomerulus. We have screened for the expression of recombinant H. influenzae IgA1 protease by combining various expression plasmids, IgA1 protease constructs, and E. coli strains under multiple conditions. Using the method we have developed, approximately 20–40 mg/L of soluble and active H. influenzae IgA1 protease can be produced from E. coli strain C41(DE3, a significant increase in yield compared to the yield upon expression in H. influenzae or other related bacteria.

  20. A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

    Science.gov (United States)

    Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

    2016-07-15

    A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

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

    Directory of Open Access Journals (Sweden)

    Lin Qu

    2011-09-01

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

  2. Dose determination in interventional procedures at a hospital, using Al2O3:C detectors

    International Nuclear Information System (INIS)

    Full text: Interventional practices have increased in most countries over the past 20 years, combining diagnostic and therapeutic procedures. However, the determination of staff doses in fluoroscopy is difficult, because the examinations are dynamic in nature. Such procedures are complex and they involve prolonged irradiations, providing high radiation doses to patients and to the staff. The effective dose of the staff can be estimated by direct methods, using the optically stimulated luminescence (OSL) technique. In the present work inLight dosimeters and an inLight microstar reader, Landauer, were utilized. These dosimeters are formed by four aluminium oxide (Al2O3:C) detectors and there are aluminium, copper and plastic filters and an open window in each badge. In this work, the dosimeters were utilized for the dose determinations in interventional procedures. Initially the calibration factors and the energy dependence of Al2O3:C dosimeters were obtained in the range of 60 to 120 kV. The doses were evaluated at different positions on the staff during the routine procedures at the Hospital Sao Paulo. The results will be compared with those previously obtained using thermoluminescent CaSO4:Dy

  3. 3C 33: another case of photoionized soft X-ray emission in radio galaxies

    CERN Document Server

    Torresi, E; Guainazzi, M; Palumbo, G G C; Ponti, G; Bianchi, S

    2009-01-01

    All the observations available in the Chandra and XMM-Newton archives have been used to investigate the X-ray spectral properties of 3C 33. In this paper is presented a complete X-ray analysis of the nuclear emission of this narrow line radio galaxy. The broad band spectrum of 3C 33 is complex. The hard part resembles that of Seyfert 2 galaxies, with a heavily obscured nuclear continuum (N_H~10^23 cm^-2) and a prominent Fe Kalpha line. This represents the nuclear radiation directly observed in transmission through a cold circumnuclear gas. On the other hand an unabsorbed continuum plus emission lines seem to fit well the soft part of the spectrum (0.5-2 keV) suggesting that the jet does not significantly contribute to the X-ray emission. We discuss the possible collisional or photoionized origin of the gas that emits the soft X-ray lines. Our results, strengthened by optical spectroscopy favor the photoionization scenario.

  4. Comparative Study on the Protease Inhibitors from Fish Eggs

    Institute of Scientific and Technical Information of China (English)

    Ustadi; K.Y.Kim; S.M.Kim

    2005-01-01

    The protease inhibitor was purified from five different fish eggs. The molecular weights of Pacific herring, chum salmon, pond smelt, glassfish, and Alaska pollock egg protease inhibitors were 120, 89, 84.5, 17, and 16.8kDa, respectively. The specific inhibitory activity of glassfish egg protease inhibitor was the highest followed by those of Pacific herring and Alaska pollock in order. The specific inhibitory activity and purity of glassfish egg protease inhibitor were 19.70 U mg-1 protein and 164.70 folds of purification, respectively. Glassfish egg protease inhibitor was reasonably stable at 50 - 65 ℃ and pH 8,which was more stable at high temperature and pH than protease inhibitors from the other fish species. Glassfish egg protease inhibitor was noncompetitive with inhibitor constant (Ki) of 4.44 nmol L-1.

  5. New directions for protease inhibitors directed drug discovery.

    Science.gov (United States)

    Hamada, Yoshio; Kiso, Yoshiaki

    2016-11-01

    Proteases play crucial roles in various biological processes, and their activities are essential for all living organisms-from viruses to humans. Since their functions are closely associated with many pathogenic mechanisms, their inhibitors or activators are important molecular targets for developing treatments for various diseases. Here, we describe drugs/drug candidates that target proteases, such as malarial plasmepsins, β-secretase, virus proteases, and dipeptidyl peptidase-4. Previously, we reported inhibitors of aspartic proteases, such as renin, human immunodeficiency virus type 1 protease, human T-lymphotropic virus type I protease, plasmepsins, and β-secretase, as drug candidates for hypertension, adult T-cell leukaemia, human T-lymphotropic virus type I-associated myelopathy, malaria, and Alzheimer's disease. Our inhibitors are also described in this review article as examples of drugs that target proteases. © 2015 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 106: 563-579, 2016. PMID:26584340

  6. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    Energy Technology Data Exchange (ETDEWEB)

    Yedidi, Ravikiran S. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Muhuhi, Joseck M. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Liu, Zhigang [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Bencze, Krisztina Z. [Department of Chemistry, Fort Hays State University, Hays, KS 67601 (United States); Koupparis, Kyriacos [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); O’Connor, Carrie E.; Kovari, Iulia A. [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States); Spaller, Mark R. [Department of Chemistry, Wayne State University, Detroit, MI 48202 (United States); Kovari, Ladislau C., E-mail: kovari@med.wayne.edu [Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201 (United States)

    2013-09-06

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC{sub 50}: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the {sup 15}N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC{sub 50}: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of {sup 15}N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV.

  7. Design, synthesis and evaluation of a potent substrate analog inhibitor identified by scanning Ala/Phe mutagenesis, mimicking substrate co-evolution, against multidrug-resistant HIV-1 protease

    International Nuclear Information System (INIS)

    Highlights: •Inhibitors against MDR HIV-1 protease were designed, synthesized and evaluated. •Lead peptide (6a) showed potent inhibition (IC50: 4.4 nM) of MDR HIV-1 protease. •(6a) Showed favorable binding isotherms against NL4-3 and MDR proteases. •(6a) Induced perturbations in the 15N-HSQC spectrum of MDR HIV-1 protease. •Molecular modeling suggested that (6a) may induce total flap closure inMDR protease. -- Abstract: Multidrug-resistant (MDR) clinical isolate-769, human immunodeficiency virus type-1 (HIV-1) protease (PDB ID: (1TW7)), was shown to exhibit wide-open flaps and an expanded active site cavity, causing loss of contacts with protease inhibitors. In the current study, the expanded active site cavity of MDR769 HIV-1 protease was screened with a series of peptide-inhibitors that were designed to mimic the natural substrate cleavage site, capsid/p2. Scanning Ala/Phe chemical mutagenesis approach was incorporated into the design of the peptide series to mimic the substrate co-evolution. Among the peptides synthesized and evaluated, a lead peptide (6a) with potent activity (IC50: 4.4 nM) was identified against the MDR769 HIV-1 protease. Isothermal titration calorimetry data showed favorable binding profile for 6aagainst both wild type and MDR769 HIV-1 protease variants. Nuclear magnetic resonance spectrum of 15N-labeled MDR769 HIV-1 protease in complex with 6a showed some major perturbations in chemical shift, supporting the peptide induced conformational changes in protease. Modeling analysis revealed multiple contacts between 6a and MDR769 HIV-1 protease. The lead peptide-inhibitor, 6a, with high potency and good binding profile can be used as the basis for developing potent small molecule inhibitors against MDR variants of HIV

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

    Science.gov (United States)

    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

    2015-07-01

    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.

  9. Synthesis and biological evaluation of nucleobase-modified analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd)

    DEFF Research Database (Denmark)

    Hrdlicka, Patrick J; Jepsen, Jan S; Nielsen, Claus;

    2005-01-01

    A series of nucleobase-modified analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd) were designed to overcome the strict substrate specificity of the activating uridine-cytidine kinase. EUrd, ECyd and target nucleosides were obtained using a short...

  10. Multi-Frequency VLBA Studies of the Parsec-Scale Jets in 3C 66A and 3C 66B

    Indian Academy of Sciences (India)

    G.-Y. Zhao; Y.-J. Chen; Z.-Q. Shen; H. Sudou; S. Iguchi; F. Gao; Y. Murata; Y. Taniguchi

    2014-09-01

    We report multi-frequency VLBA phase-referencing observation results of 3C 66A and 3C 66B, including high resolution maps and relative position measurements. The resulting images show similar morphology with that presented in previous works. We find core shift variations in both sources, indicating some physical condition changes in the jets.

  11. A Two-Dimensional Zirconium Carbide by Selective Etching of Al3C3 from Nanolaminated Zr3Al3C5.

    Science.gov (United States)

    Zhou, Jie; Zha, Xianhu; Chen, Fan Y; Ye, Qun; Eklund, Per; Du, Shiyu; Huang, Qing

    2016-04-11

    The room-temperature synthesis of a new two-dimensional (2D) zirconium-containing carbide, Zr3C2T(z) MXene is presented. In contrast to traditional preparation of MXene, the layered ternary Zr3Al3C5 material instead of MAX phases is used as source under hydrofluoric acid treatment. The structural, mechanical, and electronic properties of the synthesized 2D carbide are investigated, combined with first-principles density functional calculations. A comparative study on the structrual stability of our obtained 2D Zr3C2T(z) and Ti3C2T(z) MXenes at elevated temperatures is performed. The obtained 2D Zr3C2T(z) exhibits relatively better ability to maintain 2D nature and strucural integrity compared to Ti-based Mxene. The difference in structural stability under high temperature condition is explained by a theoretical investigation on binding energy.

  12. 18 CFR 3c.3 - Reporting fraud, waste, abuse, and corruption and cooperation with official inquiries.

    Science.gov (United States)

    2010-04-01

    ... OF CONDUCT § 3c.3 Reporting fraud, waste, abuse, and corruption and cooperation with official inquiries. (a) Employees shall, in fulfilling the obligation of 5 CFR 2635.101(b)(11), report fraud, waste..., abuse, and corruption and cooperation with official inquiries. 3c.3 Section 3c.3 Conservation of...

  13. 17 CFR 270.3c-2 - Definition of beneficial ownership in small business investment companies.

    Science.gov (United States)

    2010-04-01

    ... 1940 § 270.3c-2 Definition of beneficial ownership in small business investment companies. For the... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Definition of beneficial ownership in small business investment companies. 270.3c-2 Section 270.3c-2 Commodity and...

  14. Protease Activation and Inflammation in Acute Pancreatitis

    OpenAIRE

    Regnér, Sara

    2008-01-01

    Approximately 10—20 % of patients with acute pancreatitis (AP) develop a severe disease with high mortality and morbidity. Activation of pancreatic proteases, the inflammatory response and impaired pancreatic circulation are pathophysiological events that are important in order for the disease to develop. There is no specific treatment for severe AP, and no useful marker for predicting the severity of the disease upon admission to the hospital. In this thesis, markers of early pathophysio...

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

    Science.gov (United States)

    Budatha, Madhusudhan; Silva, Simone; Montoya, Teodoro Ignacio; Suzuki, Ayako; Shah-Simpson, Sheena; Wieslander, Cecilia Karin; Yanagisawa, Masashi; Word, Ruth Ann; Yanagisawa, Hiromi

    2013-01-01

    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. PMID:23437119

  16. 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.

  17. Acanthamoeba protease activity promotes allergic airway inflammation via protease-activated receptor 2.

    Directory of Open Access Journals (Sweden)

    Mi Kyung Park

    Full Text Available Acanthamoeba is a free-living amoeba commonly present in the environment and often found in human airway cavities. Acanthamoeba possesses strong proteases that can elicit allergic airway inflammation. To our knowledge, the aeroallergenicity of Acanthamoeba has not been reported. We repeatedly inoculated mice with Acanthamoeba trophozoites or excretory-secretory (ES proteins intra-nasally and evaluated symptoms and airway immune responses. Acanthamoeba trophozoites or ES proteins elicited immune responses in mice that resembled allergic airway inflammation. ES proteins had strong protease activity and activated the expression of several chemokine genes (CCL11, CCL17, CCL22, TSLP, and IL-25 in mouse lung epithelial cells. The serine protease inhibitor phenyl-methane-sulfonyl fluoride (PMSF inhibited ES protein activity. ES proteins also stimulated dendritic cells and enhanced the differentiation of naive T cells into IL-4-secreting T cells. After repeated inoculation of the protease-activated receptor 2 knockout mouse with ES proteins, airway inflammation and Th2 immune responses were markedly reduced, but not to basal levels. Furthermore, asthma patients had higher Acanthamoeba-specific IgE titers than healthy controls and we found Acanthamoeba specific antigen from house dust in typical living room. Our findings suggest that Acanthamoeba elicits allergic airway symptoms in mice via a protease allergen. In addition, it is possible that Acanthamoeba may be one of the triggers human airway allergic disease.

  18. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    M El Bakkouri; A Pow; A Mulichak; K Cheung; J Artz; M Amani; S Fell; T de Koning-Ward; C Goodman; et al.

    2011-12-31

    The Clpchaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clpchaperones and proteases in the humanmalariaparasitePlasmodiumfalciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clpchaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  19. The Clp Chaperones and Proteases of the Human Malaria Parasite Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bakkouri, Majida El; Pow, Andre; Mulichak, Anne; Cheung, Kevin L.Y.; Artz, Jennifer D.; Amani, Mehrnaz; Fell, Stuart; de Koning-Ward, Tania F.; Goodman, C. Dean; McFadden, Geoffrey I.; Ortega, Joaquin; Hui, Raymond; Houry, Walid A. (McMaster U.); (Melbourne); (Toronto); (Deakin); (HWMRI)

    2015-02-09

    The Clp chaperones and proteases play an important role in protein homeostasis in the cell. They are highly conserved across prokaryotes and found also in the mitochondria of eukaryotes and the chloroplasts of plants. They function mainly in the disaggregation, unfolding and degradation of native as well as misfolded proteins. Here, we provide a comprehensive analysis of the Clp chaperones and proteases in the human malaria parasite Plasmodium falciparum. The parasite contains four Clp ATPases, which we term PfClpB1, PfClpB2, PfClpC and PfClpM. One PfClpP, the proteolytic subunit, and one PfClpR, which is an inactive version of the protease, were also identified. Expression of all Clp chaperones and proteases was confirmed in blood-stage parasites. The proteins were localized to the apicoplast, a non-photosynthetic organelle that accommodates several important metabolic pathways in P. falciparum, with the exception of PfClpB2 (also known as Hsp101), which was found in the parasitophorous vacuole. Both PfClpP and PfClpR form mostly homoheptameric rings as observed by size-exclusion chromatography, analytical ultracentrifugation and electron microscopy. The X-ray structure of PfClpP showed the protein as a compacted tetradecamer similar to that observed for Streptococcus pneumoniae and Mycobacterium tuberculosis ClpPs. Our data suggest the presence of a ClpCRP complex in the apicoplast of P. falciparum.

  20. 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.

  1. Protease gene shuffling and expression in Pichia pastoris

    Directory of Open Access Journals (Sweden)

    Gang Yang

    2015-06-01

    Full Text Available Four kinds of neutral and alkaline protease genes from Aspergillus oryzae and Bacillus subtilis were isolated and shuffled. The shuffled genes were selected, inserted into pGAPZαA plasmid and transformed into Escherichia coli. The gene which could express high-activity protease was selected by screening the sizes of transparent zones around the colonies on casein plates. After an ideal protease gene was selected, it was sequenced and then transformed into Pichia pastoris X33. The result showed that the base in 1022th position of shuffled protease gene was changed from thymine to cytosine, inferring that cysteine was changed to arginine in the mutant protease. After 48 h incubation for the transformed P. pastoris with the mutant or native protease genes, the mutant protease activity was 36.4% higher than the native protease (P<0.05. The optimal pH and temperature of the mutant protease were 6.5-8.0 and 30-70°C, respectively, which indicated better stability than the native protease (P<0.05.

  2. Mitochondrial Proteases as Emerging Pharmacological Targets.

    Science.gov (United States)

    Gibellini, Lara; De Biasi, Sara; Nasi, Milena; Iannone, Anna; Cossarizza, Andrea; Pinti, Marcello

    2016-01-01

    The preservation of mitochondrial function and integrity is critical for cell viability. Under stress conditions, unfolded, misfolded or damaged proteins accumulate in a certain compartment of the organelle, interfering with oxidative phosphorylation and normal mitochondrial functions. In stress conditions, several mechanisms, including mitochondrial unfolded protease response (UPRmt), fusion and fission, and mitophagy are engaged to restore normal proteostasis of the organelle. Mitochondrial proteases are a family of more than 20 enzymes that not only are involved in the UPRmt, but actively participate at multiple levels in the stress-response system. Alterations in their expression levels, or mutations that determine loss or gain of function of these proteases deeply impair mitochondrial functionality and can be associated with the onset of inherited diseases, with the development of neurodegenerative disorders and with the process of carcinogenesis. In this review, we focus our attention on six of them, namely CLPP, HTRA2 and LONP1, by analysing the current knowledge about their functions, their involvement in the pathogenesis of human diseases, and the compounds currently available for inhibiting their functions. PMID:26831646

  3. Structure and Properties of the Nonface-Spiral Fullerenes T-C380, D3-C384, D3-C440, and D3-C672 and Their Halma and Leapfrog Transforms

    DEFF Research Database (Denmark)

    Wirz, Lukas; Tonner, Ralf; Avery, James Emil;

    2013-01-01

    The structure and properties of the three smallest nonface-spiral (NS) fullerenes NS-T-C380, NS-D3-C384, NS-D3-C440, and the first isolated pentagon NS-fullerene, NS-D3-C672, are investigated in detail. They are constructed by either a generalized face-spiral algorithm or by vertex insertions...... followed by a force-field optimization using the recently introduced program Fullerene. The obtained structures were then further optimized at the density functional level of theory and their stability analyzed with reference to Ih-C60. The large number of hexagons results in a higher stability of the NS-fullerenes...... compared to C60, but, as expected, in a lower stability than most stable isomers. None of the many investigated halma transforms on nonspiral fullerenes, NS-T-C380, NS-D3-C384, NS-D3-C440, and NS-D3-C672, admit any spirals, and we conjecture that all halma transforms of NS-fullerenes belong to the class...

  4. The nuclear protein Sam68 is cleaved by the FMDV 3C protease redistributing Sam68 to the cytoplasm during FMDV infection of host cells

    International Nuclear Information System (INIS)

    Picornavirus infection can lead to disruption of nuclear pore traffic, shut-off of cell translation machinery, and cleavage of proteins involved in cellular signal transduction and the innate response to infection. Here, we demonstrated that the FMDV 3Cpro induced the cleavage of nuclear RNA-binding protein Sam68 C-terminus containing the nuclear localization sequence (NLS). Consequently, it stimulated the redistribution of Sam68 to the cytoplasm. The siRNA knockdown of Sam68 resulted in a 1000-fold reduction in viral titers, which prompted us to study the effect of Sam68 on FMDV post-entry events. Interestingly, Sam68 interacts with the internal ribosomal entry site within the 5′ non-translated region of the FMDV genome, and Sam68 knockdown decreased FMDV IRES-driven activity in vitro suggesting that it could modulate translation of the viral genome. The results uncover a novel role for Sam68 in the context of picornaviruses and the proteolysis of a new cellular target of the FMDV 3Cpro.

  5. Efficient production of foot-and-mouth disease virus empty capsids in insect cells following down regulation of 3C protease activity

    DEFF Research Database (Denmark)

    Porta, Claudine; Xu, Xiaodong; Loureiro, Silvia;

    2013-01-01

    Foot-and-mouth disease virus (FMDV) is a significant economically and distributed globally pathogen of Artiodactyla. Current vaccines are chemically inactivated whole virus particles that require large-scale virus growth in strict bio-containment with the associated risks of accidental release or...

  6. Critical clamp loader processing by an essential AAA+ protease in Caulobacter crescentus.

    Science.gov (United States)

    Vass, Robert H; Chien, Peter

    2013-11-01

    Chromosome replication relies on sliding clamps that are loaded by energy-dependent complexes. In Escherichia coli, the ATP-binding clamp loader subunit DnaX exists as both long (τ) and short (γ) forms generated through programmed translational frameshifting, but the need for both forms is unclear. Here, we show that in Caulobacter crescentus, DnaX isoforms are unexpectedly generated through partial proteolysis by the AAA+ protease casein lytic proteinase (Clp) XP. We find that the normally processive ClpXP protease partially degrades DnaX to produce stable fragments upon encountering a glycine-rich region adjacent to a structured domain. Increasing the sequence complexity of this region prevents partial proteolysis and generates a τ-only form of DnaX in vivo that is unable to support viability on its own. Growth is restored when γ is provided in trans, but these strains are more sensitive to DNA damage compared with strains that can generate γ through proteolysis. Our work reveals an unexpected mode of partial processing by the ClpXP protease to generate DnaX isoforms, demonstrates that both τ and γ forms of DnaX are required for Caulobacter viability, and identifies a role for clamp loader diversity in responding to DNA damage. The conservation of distinct DnaX isoforms throughout bacteria despite fundamentally different mechanisms for producing them suggests there may be a conserved need for alternate clamp loader complexes during DNA damaging conditions.

  7. A preliminary neutron diffraction analysis of Achromobacter protease I

    Science.gov (United States)

    Ohnishi, Yuki; Masaki, Takeharu; Yamada, Taro; Kurihara, Kazuo; Tanaka, Ichiro; Niimura, Nobuo

    2010-11-01

    Achromobacter protease I (API, E.C. 3.4.21.50) is one of the serine proteases produced by Achromobacter lyticus M497-1. API is distinct from the other tripsin type protease in its lysine specificity. The neutron structure analysis of catalytic triad with Trp169 and His210 was presented. His57 was double protonated and formed hydrogen bonds to Ser194Oγ and Asp113Oδ1, Oδ2.

  8. Hydrophobic core flexibility modulates enzyme activity in HIV-1 protease

    OpenAIRE

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.; Bolon, Daniel N. A.; Schiffer, Celia A.

    2012-01-01

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Di...

  9. Enhanced Thermostability of a Fungal Alkaline Protease by Different Additives

    OpenAIRE

    Nilesh P. Nirmal; R. Seeta Laxman

    2014-01-01

    A fungal strain (Conidiobolus brefeldianus MTCC 5184) isolated from plant detritus secreted a high activity alkaline protease. Thermostability studies of the fungal alkaline protease (FAP) revealed that the protease is stable up to 50°C with 40% residual activity after one hour. Effect of various additives such as sugars, sugar alcohols, polyols, and salts, on the thermostability of FAP was evaluated. Among the additives tested, glycerol, mannitol, xylitol, sorbitol, and trehalose were found ...

  10. Heighten the Study on Factor Seven Activating Protease

    Institute of Scientific and Technical Information of China (English)

    贺石林; 陈方平; 张广森; 文志斌

    2008-01-01

    @@ Recent studies have showed that factor seven activating protease (FSAP) is a novel serine protease in human plasma. Immunoreactivity for FSAP has been observed in vascular endothelial cells,epithelial cells and macrophages but FSAP-specific mRNA expression only exists in the former two cells. FSAP has three epidermal growth factor (EGF) domains,a kringle domain and a serine protease domain.

  11. GADD45A inhibits autophagy by regulating the interaction between BECN1 and PIK3C3.

    Science.gov (United States)

    Zhang, Dongdong; Zhang, Weimin; Li, Dan; Fu, Ming; Chen, Runsheng; Zhan, Qimin

    2015-01-01

    GADD45A is a TP53-regulated and DNA damage-inducible tumor suppressor protein, which regulates cell cycle arrest, apoptosis, and DNA repair, and inhibits tumor growth and angiogenesis. However, the function of GADD45A in autophagy remains unknown. In this report, we demonstrate that GADD45A plays an important role in regulating the process of autophagy. GADD45A is able to decrease LC3-II expression and numbers of autophagosomes in mouse tissues and different cancer cell lines. Using bafilomycin A1 treatment, we have observed that GADD45A regulates autophagosome initiation. Likely, GADD45A inhibition of autophagy is through its influence on the interaction between BECN1 and PIK3C3. Immunoprecipitation and GST affinity isolation assays exhibit that GADD45A directly interacts with BECN1, and in turn dissociates the BECN1-PIK3C3 complex. Furthermore, we have mapped the 71 to 81 amino acids of the GADD45A protein that are necessary for the GADD45A interaction with BECN1. Knockdown of BECN1 can abolish autophagy alterations induced by GADD45A. Taken together, these findings provide the novel evidence that GADD45A inhibits autophagy via impairing the BECN1-PIK3C3 complex formation.

  12. Probing the Disk-Jet Connection of the Radio Galaxy 3C120 Observed With Suzaku

    Energy Technology Data Exchange (ETDEWEB)

    Kataoka, Jun; Reeves, James N.; Iwasawa, Kazushi; Markowitz, Alex G.; Mushotzky, Richard F.; Arimoto, Makoto; Takahashi, Tadayuki; Tsubuku, Yoshihiro; Ushio, Masayoshi; Watanabe, Shin; Gallo, Luigi C.; Madejski, Greg M.; Terashima, Yuichi; Isobe, Naoki; Tashiro, Makoto S.; Kohmura, Takayoshi; /Tokyo Inst. Tech. /NASA, Goddard /Garching, Max Planck

    2007-01-03

    Broad line radio galaxies (BLRGs) are a rare type of radio-loud AGN, in which the broad optical permitted emission lines have been detected in addition to the extended jet emission. Here we report on deep (40ksec x 4) observations of the bright BLRG 3C 120 using Suzaku. The observations were spaced a week apart, and sample a range of continuum fluxes. An excellent broadband spectrum was obtained over two decades of frequency (0.6 to 50 keV) within each 40 ksec exposure. We clearly resolved the iron K emission line complex, finding that it consists of a narrow K{sub {alpha}} core ({sigma} {approx_equal} 110 eV or an EW of 60 eV), a 6.9 keV line, and an underlying broad iron line. Our confirmation of the broad line contrasts with the XMM-Newton observation in 2003, where the broad line was not required. The most natural interpretation of the broad line is iron K line emission from a face-on accretion disk which is truncated at {approx} 10 r{sub g}. Above 10 keV, a relatively weak Compton hump was detected (reflection fraction of R {approx_equal} 0.6), superposed on the primary X-ray continuum of {Lambda} {approx_equal} 1.75. Thanks to the good photon statistics and low background of the Suzaku data, we clearly confirm the spectral evolution of 3C 120, whereby the variability amplitude decreases with increasing energy. More strikingly, we discovered that the variability is caused by a steep power-law component of {Lambda} {approx_equal} 2.7, possibly related to the non-thermal jet emission. We discuss our findings in the context of similarities and differences between radio-loud/quiet objects.

  13. Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

    Science.gov (United States)

    Petersen, Lauren M; Tisa, Louis S

    2014-11-01

    A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. PMID:25182493

  14. The High Energy view of the Broad Line Radio Galaxy 3C 111

    CERN Document Server

    Ballo, L; Reeves, J N; Sambruna, R M; Tombesi, F

    2011-01-01

    We present the analysis of Suzaku and XMM-Newton observations of the broad-line radio galaxy (BLRG) 3C 111. Its high energy emission shows variability, a harder continuum with respect to the radio quiet AGN population, and weak reflection features. Suzaku found the source in a minimum flux level; a comparison with the XMM-Newton data implies an increase of a factor of 2.5 in the 0.5-10 keV flux, in the 6 months separating the two observations. The iron K complex is detected in both datasets, with rather low equivalent width(s). The intensity of the iron K complex does not respond to the change in continuum flux. An ultra-fast, high-ionization outflowing gas is clearly detected in the XIS data; the absorber is most likely unstable. Indeed, during the XMM-Newton observation, which was 6 months after, the absorber was not detected. No clear roll-over in the hard X-ray emission is detected, probably due to the emergence of the jet as a dominant component in the hard X-ray band, as suggested by the detection above...

  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. Role of Protease-Inhibitors in Ocular Diseases.

    Science.gov (United States)

    Pescosolido, Nicola; Barbato, Andrea; Pascarella, Antonia; Giannotti, Rossella; Genzano, Martina; Nebbioso, Marcella

    2014-01-01

    It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI), metalloproteinase inhibitor (TIMP), maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI), and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models. PMID:25493637

  17. Role of Protease-Inhibitors in Ocular Diseases

    Directory of Open Access Journals (Sweden)

    Nicola Pescosolido

    2014-12-01

    Full Text Available It has been demonstrated that the balance between proteases and protease-inhibitors system plays a key role in maintaining cellular and tissue homeostasis. Indeed, its alteration has been involved in many ocular and systemic diseases. In particular, research has focused on keratoconus, corneal wounds and ulcers, keratitis, endophthalmitis, age-related macular degeneration, Sorsby fundus dystrophy, loss of nerve cells and photoreceptors during optic neuritis both in vivo and in vitro models. Protease-inhibitors have been extensively studied, rather than proteases, because they may represent a therapeutic approach for some ocular diseases. The protease-inhibitors mainly involved in the onset of the above-mentioned ocular pathologies are: α2-macroglobulin, α1-proteinase inhibitor (α1-PI, metalloproteinase inhibitor (TIMP, maspin, SERPINA3K, SERPINB13, secretory leukocyte protease inhibitor (SLPI, and calpeptin. This review is focused on the several characteristics of dysregulation of this system and, particularly, on a possible role of proteases and protease-inhibitors in molecular remodeling that may lead to some ocular diseases. Recently, researchers have even hypothesized a possible therapeutic effect of the protease-inhibitors in the treatment of injured eye in animal models.

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

    International Nuclear Information System (INIS)

    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. 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.

  20. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    Science.gov (United States)

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies.

  1. EBV Nuclear Antigen 3C Mediates Regulation of E2F6 to Inhibit E2F1 Transcription and Promote Cell Proliferation.

    Science.gov (United States)

    Pei, Yonggang; Banerjee, Shuvomoy; Sun, Zhiguo; Jha, Hem Chandra; Saha, Abhik; Robertson, Erle S

    2016-08-01

    Epstein-Barr virus (EBV) is considered a ubiquitous herpesvirus with the ability to cause latent infection in humans worldwide. EBV-association is evidently linked to different types of human malignancies, mainly of epithelial and lymphoid origin. Of interest is the EBV nuclear antigen 3C (EBNA3C) which is critical for EBV-mediated immortalization. Recently, EBNA3C was shown to bind the E2F1 transcription regulator. The E2F transcription factors have crucial roles in various cellular functions, including cell cycle, DNA replication, DNA repair, cell mitosis, and cell fate. Specifically, E2F6, one of the unique E2F family members, is known to be a pRb-independent transcription repressor of E2F-target genes. In our current study, we explore the role of EBNA3C in regulating E2F6 activities. We observed that EBNA3C plays an important role in inducing E2F6 expression in LCLs. Our study also shows that EBNA3C physically interacts with E2F6 at its amino and carboxy terminal domains and they form a protein complex in human cells. In addition, EBNA3C stabilizes the E2F6 protein and is co-localized in the nucleus. We also demonstrated that both EBNA3C and E2F6 contribute to reduction in E2F1 transcriptional activity. Moreover, E2F1 forms a protein complex with EBNA3C and E2F6, and EBNA3C competes with E2F1 for E2F6 binding. E2F6 is also recruited by EBNA3C to the E2F1 promoter, which is critical for EBNA3C-mediated cell proliferation. These results demonstrate a critical role for E2F family members in EBV-induced malignancies, and provide new insights for targeting E2F transcription factors in EBV-associated cancers as potential therapeutic intervention strategies. PMID:27548379

  2. Primary structural analysis of sulfhydryl protease inhibitors from pineapple stem.

    Science.gov (United States)

    Reddy, M N; Keim, P S; Heinrikson, R L; Kezdy, F J

    1975-03-10

    Pineapple stem acetone powder provides a rich source of the sulfhydryl protease bromelain and of a family of compositionally similar but chromatographically distinct polypeptide inihibtors of this enzyme. The isoinhibitors have molecular weights of 5600, and they contain five disulfide bonds and about 50 amino acids each (Perlstein, S. H., AND Kezdy, F.J. (1973) J. Supramol. Struct. 1, 249-254). Primary structural analysis of one of the seven inhibitor fractions (VII) revealed extensive microheterogeneity. Each of the inhibitor molecules in Fraction VII was shown to be composed of two peptide chains joined by disulfide bonds. These chains, designated A and B on the basis of size, comprise 41 and 10-11 residues, respectively, and the amino acid sequence of one of each are given below: (see article for formular). On the basis of ionization properties and yields of the A and B chains, it would appear that one of the major inhibitor species in Fraction VII is the covalently linked complex of the two chains shown, namely [A-1, B-2]. The second major inhibitor component of Fraction VII is identical in structure with [A-1, B-2i1 except that residues 1 and 8 in the A chain are pyroglutamate and threonine, respectively, and in the B chain glutamine 11 is replaced by arginine. The third inhibitor in Fraction VII is a minor constituent identical with the second, except that residue 1 in the A chain is glutamate rather than pyroglutamate. This microheterogeneity in the inhibitors of Fraction VII is further increased by the fact that B chains may lack threonine 1, in which case they are decapeptides beginning with alanine. On the basis of the striking homology of the cysteine residues with those of other protease inhibitors, it is proposed that the bromelain inhibitors are generated enzymatically from single chain precursors by excision of a "bridge" paptide which links the NH-2 termal A chain to the COOH-terminal B chain.

  3. Synthesis and biological evaluation of branched and conformationally restricted analogs of the anticancer compounds 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd)

    DEFF Research Database (Denmark)

    Hrdlicka, Patrick J; Andersen, Nicolai K; Jepsen, Jan S;

    2005-01-01

    The synthesis of branched and conformationally restricted analogs of the anticancer nucleosides 3'-C-ethynyluridine (EUrd) and 3'-C-ethynylcytidine (ECyd) is presented. Molecular modeling and (1)H NMR coupling constant analysis revealed that the furanose rings of all analogs except the LNA analog...... are conformationally biased towards South conformation, and are thus mimicking the structure of ECyd. All target nucleosides were devoid of anti-HIV or anticancer activity....

  4. Structural Insights into the Activation and Inhibition of Histo-Aspartic Protease from Plasmodium falciparum

    Energy Technology Data Exchange (ETDEWEB)

    Bhaumik, Prasenjit; Xiao, Huogen; Hidaka, Koushi; Gustchina, Alla; Kiso, Yoshiaki; Yada, Rickey Y.; Wlodawer, Alexander (Guelph); (Kyoto); (NCI)

    2012-09-17

    Histo-aspartic protease (HAP) from Plasmodium falciparum is a promising target for the development of novel antimalarial drugs. The sequence of HAP is highly similar to those of pepsin-like aspartic proteases, but one of the two catalytic aspartates, Asp32, is replaced with histidine. Crystal structures of the truncated zymogen of HAP and of the complex of the mature enzyme with inhibitor KNI-10395 have been determined at 2.1 and 2.5 {angstrom} resolution, respectively. As in other proplasmepsins, the propeptide of the zymogen interacts with the C-terminal domain of the enzyme, forcing the N- and C-terminal domains apart, thereby separating His32 and Asp215 and preventing formation of the mature active site. In the inhibitor complex, the enzyme forms a tight domain-swapped dimer, not previously seen in any aspartic proteases. The inhibitor is found in an unprecedented conformation resembling the letter U, stabilized by two intramolecular hydrogen bonds. Surprisingly, the location and conformation of the inhibitor are similar to those of the fragment of helix 2 comprising residues 34p-38p in the prosegments of the zymogens of gastric aspartic proteases; a corresponding helix assumes a vastly different orientation in proplasmepsins. Each inhibitor molecule is in contact with two molecules of HAP, interacting with the carboxylate group of the catalytic Asp215 of one HAP protomer through a water molecule, while also making a direct hydrogen bond to Glu278A' of the other protomer. A comparison of the shifts in the positions of the catalytic residues in the inhibitor complex presented here with those published previously gives further hints regarding the enzymatic mechanism of HAP.

  5. CVD growth and properties of boron phosphide on 3C-SiC

    Science.gov (United States)

    Padavala, Balabalaji; Frye, C. D.; Wang, Xuejing; Raghothamachar, Balaji; Edgar, J. H.

    2016-09-01

    Improving the crystalline quality of boron phosphide (BP) is essential for realizing its full potential in semiconductor device applications. In this study, 3C-SiC was tested as a substrate for BP epitaxy. BP films were grown on 3C-SiC(100)/Si, 3C-SiC(111)/Si, and 3C-SiC(111)/4H-SiC(0001) substrates in a horizontal chemical vapor deposition (CVD) system. Films were produced with good crystalline orientation and morphological features in the temperature range of 1000-1200 °C using a PH3+B2H6+H2 mixture. Rotational twinning was absent in the BP due to the crystal symmetry-matching with 3C-SiC. Confocal 3D Raman imaging of BP films revealed primarily uniform peak shift and peak widths across the scanned area, except at defects on the surface. Synchrotron white beam X-ray topography showed the epitaxial relationship between BP and 3C-SiC was (100) BP||(100) 3C-SiC and (111) BP||(111) 3C-SiC. Scanning electron microscopy, Raman spectroscopy and X-ray diffraction analysis indicated residual tensile strain in the films and improved crystalline quality at temperatures below 1200 °C. These results indicated that BP properties could be further enhanced by employing high quality bulk 3C-SiC or 3C-SiC epilayers on 4H-SiC substrates.

  6. The SARS-coronavirus papain-like protease: structure, function and inhibition by designed antiviral compounds.

    Science.gov (United States)

    Báez-Santos, Yahira M; St John, Sarah E; Mesecar, Andrew D

    2015-03-01

    Over 10 years have passed since the deadly human coronavirus that causes severe acute respiratory syndrome (SARS-CoV) emerged from the Guangdong Province of China. Despite the fact that the SARS-CoV pandemic infected over 8500 individuals, claimed over 800 lives and cost billions of dollars in economic loss worldwide, there still are no clinically approved antiviral drugs, vaccines or monoclonal antibody therapies to treat SARS-CoV infections. The recent emergence of the deadly human coronavirus that causes Middle East respiratory syndrome (MERS-CoV) is a sobering reminder that new and deadly coronaviruses can emerge at any time with the potential to become pandemics. Therefore, the continued development of therapeutic and prophylactic countermeasures to potentially deadly coronaviruses is warranted. The coronaviral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro), are attractive antiviral drug targets because they are essential for coronaviral replication. Although the primary function of PLpro and 3CLpro are to process the viral polyprotein in a coordinated manner, PLpro has the additional function of stripping ubiquitin and ISG15 from host-cell proteins to aid coronaviruses in their evasion of the host innate immune responses. Therefore, targeting PLpro with antiviral drugs may have an advantage in not only inhibiting viral replication but also inhibiting the dysregulation of signaling cascades in infected cells that may lead to cell death in surrounding, uninfected cells. This review provides an up-to-date discussion on the SARS-CoV papain-like protease including a brief overview of the SARS-CoV genome and replication followed by a more in-depth discussion on the structure and catalytic mechanism of SARS-CoV PLpro, the multiple cellular functions of SARS-CoV PLpro, the inhibition of SARS-CoV PLpro by small molecule inhibitors, and the prospect of inhibiting papain-like protease from other coronaviruses. This paper forms part of a series of

  7. 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.

  8. Rigidity analysis of HIV-1 protease

    Energy Technology Data Exchange (ETDEWEB)

    Heal, J W [MOAC Doctoral Training Centre, University of Warwick, Coventry, CV4 7AL (United Kingdom); Wells, S A; Jimenez-Roldan, E; Roemer, R A [Department of Physics and Centre for Scientific Computing, University of Warwick, Coventry, CV4 7AL (United Kingdom); Freedman, R F, E-mail: jack.heal@warwick.ac.uk [School of Life Sciences, University of Warwick, Coventry, CV4 7AL (United Kingdom)

    2011-03-01

    We present a rigidity analysis on a large number of X-ray crystal structures of the enzyme HIV-1 protease using the 'pebble game' algorithm of the software FIRST. We find that although the rigidity profile remains similar across a comprehensive set of high resolution structures, the profile changes significantly in the presence of an inhibitor. Our study shows that the action of the inhibitors is to restrict the flexibility of the {beta}-hairpin flaps which allow access to the active site. The results are discussed in the context of full molecular dynamics simulations as well as data from NMR experiments.

  9. Cathepsin F cysteine protease of the human liver fluke, Opisthorchis viverrini.

    Directory of Open Access Journals (Sweden)

    Porntip Pinlaor

    Full Text Available BACKGROUND: The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities. METHODOLOGY/PRINCIPAL FINDINGS: Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa, prosegment (95 aa, and mature protease (213 aa. BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%, Paragonimus westermani (58%, Schistosoma mansoni and S. japonicum (52%, and with vertebrate cathepsin F (51%. Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant

  10. Isolation of a thiol-dependent serine protease in peanut and investigation of its role in the complement and the allergic reaction.

    Science.gov (United States)

    Javaux, Cédric; Stordeur, Patrick; Azarkan, Mohamed; Mascart, Françoise; Baeyens-Volant, Danielle

    2016-07-01

    A serine protease activity was detected in aqueous peanuts seeds extracts, partially purified and characterized as a thiol-dependent serine protease. The potential role of this proteolytic activity on allergic reaction to peanuts was prospected through complement activation studies in human plasma and serum, and MDCK cells to investigate a possible occludin degradation in tight junctions. The peanut protease activity induced the production of anaphylatoxins C3a and C5a, and of the terminal membrane attack complex SC5b-9 whatever the complement activation pathway. The protease activity was also involved in the partial digestion of occludin within tight junctions, with for result, an increase of the epithelial permeability to antigen absorption. PMID:27280846

  11. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice

    Science.gov (United States)

    Gao, Peng

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

  12. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice.

    Science.gov (United States)

    Xie, Yinli; Gao, Peng; Li, Zhiyong

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expressed in replicative-deficient human adenovirus type 5 vector. BALB/c mice immunized intramuscularly and intraperitoneally with recombinant adenovirus rAdv-P12A3C elicited higher FMDV-specific IgG antibodies, IFN-γ, and IL-4 cytokines than those in mice immunized with inactivated FMDV vaccine. Moreover, BALB/c mice immunized with recombinant adenovirus rAdv-P12A3C by oral and intraocular-nasal immunization induced high FMDV-specific IgA antibodies. These results show that the recombinant adenovirus rAdv-P12A3C could resist FMDV comprehensively. This study highlights the potential of rAdv-P12A3C to serve as a type O FMDV vaccine. PMID:27478836

  13. A Recombinant Adenovirus Expressing P12A and 3C Protein of the Type O Foot-and-Mouth Disease Virus Stimulates Systemic and Mucosal Immune Responses in Mice

    OpenAIRE

    Yinli Xie; Peng Gao; Zhiyong Li

    2016-01-01

    Foot-and-mouth disease (FMD) is a highly contagious livestock disease of cloven-hoofed animals which causes severe economic losses. The replication-deficient, human adenovirus-vectored FMD vaccine has been proven effective against FMD. However, the role of T-cell-mediated antiviral responses and the mucosae-mediated antiviral responses induced by the adenovirus-vectored FMD vaccine was rarely examined. Here, the capsid protein precursor P1-2A and viral protease 3C of the type O FMDV were expr...

  14. Substrate profiling of tobacco etch virus protease using a novel fluorescence-assisted whole-cell assay.

    Directory of Open Access Journals (Sweden)

    George Kostallas

    Full Text Available Site-specific proteolysis of proteins plays an important role in many cellular functions and is often key to the virulence of infectious organisms. Efficient methods for characterization of proteases and their substrates will therefore help us understand these fundamental processes and thereby hopefully point towards new therapeutic strategies. Here, a novel whole-cell in vivo method was used to investigate the substrate preference of the sequence specific tobacco etch virus protease (TEVp. The assay, which utilizes protease-mediated intracellular rescue of genetically encoded short-lived fluorescent substrate reporters to enhance the fluorescence of the entire cell, allowed subtle differences in the processing efficiency of closely related substrate peptides to be detected. Quantitative screening of large combinatorial substrate libraries, through flow cytometry analysis and cell sorting, enabled identification of optimal substrates for TEVp. The peptide, ENLYFQG, identical to the protease's natural substrate peptide, emerged as a strong consensus cleavage sequence, and position P3 (tyrosine, Y and P1 (glutamine, Q within the substrate peptide were confirmed as being the most important specificity determinants. In position P1', glycine (G, serine (S, cysteine (C, alanine (A and arginine (R were among the most prevalent residues observed, all known to generate functional TEVp substrates and largely in line with other published studies stating that there is a strong preference for short aliphatic residues in this position. Interestingly, given the complex hydrogen-bonding network that the P6 glutamate (E is engaged in within the substrate-enzyme complex, an unexpectedly relaxed residue preference was revealed for this position, which has not been reported earlier. Thus, in the light of our results, we believe that our assay, besides enabling protease substrate profiling, also may serve as a highly competitive platform for directed evolution of

  15. X-Ray Photoelectron Spectrum Analysis of Yb3C60 Compound

    Institute of Scientific and Technical Information of China (English)

    CAO Xue-Wei; SHAO Yue; WANG Yu-Fang; LAN Guo-Xiang

    2001-01-01

    The nominal composition of the Yb3 C60 compound is characterized by means of x-ray photoelectron spectroscopy.Evidence of the divalent state for the Yb cation in the as-grown crystalline Yb3C60 is obtained. After exposure to air, the Yb3C60 compound transforms to an amorphous phase and Yb2O3 compound, while the valence state of the Yb cations changes from divalent to trivalent.

  16. Loss of the SUMO protease Ulp2 triggers a specific multichromosome aneuploidy.

    Science.gov (United States)

    Ryu, Hong-Yeoul; Wilson, Nicole R; Mehta, Sameet; Hwang, Soo Seok; Hochstrasser, Mark

    2016-08-15

    Post-translational protein modification by the small ubiquitin-related modifier (SUMO) regulates numerous cellular pathways, including transcription, cell division, and genome maintenance. The SUMO protease Ulp2 modulates many of these SUMO-dependent processes in budding yeast. From whole-genome RNA sequencing (RNA-seq), we unexpectedly discovered that cells lacking Ulp2 display a twofold increase in transcript levels across two particular chromosomes: chromosome I (ChrI) and ChrXII. This is due to the two chromosomes being present at twice their normal copy number. An abnormal number of chromosomes, termed aneuploidy, is usually deleterious. However, development of specific aneuploidies allows rapid adaptation to cellular stresses, and aneuploidy characterizes most human tumors. Extra copies of ChrI and ChrXII appear quickly following loss of active Ulp2 and can be eliminated following reintroduction of ULP2, suggesting that aneuploidy is a reversible adaptive mechanism to counteract loss of the SUMO protease. Importantly, increased dosage of two genes on ChrI-CLN3 and CCR4, encoding a G1-phase cyclin and a subunit of the Ccr4-Not deadenylase complex, respectively-suppresses ulp2Δ aneuploidy, suggesting that increased levels of these genes underlie the aneuploidy induced by Ulp2 loss. Our results reveal a complex aneuploidy mechanism that adapts cells to loss of the SUMO protease Ulp2. PMID:27585592

  17. A review on production of serine alkaline protease by Bacillus spp

    OpenAIRE

    Biswanath Bhunia; Bikram Basak; Apurba Dey

    2012-01-01

    In recent times, protease has gained considerable importance in the world market. Proteases are groups of proteins included in the subclass hydrolases, within the main class enzymes. Serine alkaline proteases (SAP) are one of the most important groups of industrial enzymes. They account for approximately 35% of the total microbial enzyme sales. Serine protease is produced by various types of fermentation techniques using microorganism. Among the proteases, bacterial proteases are more signifi...

  18. 3C-SiC/ZnS heterostructured nanospheres with high photocatalytic activity and enhancement mechanism

    International Nuclear Information System (INIS)

    3C-SiC/n-type ZnS heterostructured nanospheres synthesized hydrothermally deliver enhanced photocatalytic performance under visible light excitation. The heterostructured catalysts consisting of 3C-SiC and ZnS nanocrystals with a mean size being less than 5 nm exhibit extended light absorption to the visible range. The proper band structure of the 3C-SiC and ZnS nanocrystals and intrinsic electric field induced by the heterojunction promote separation of photoexcited electrons and holes in the ZnS and 3C-SiC nanocrystals resulting in the increased photocatalytic efficiency. The associated mechanism is studied and proposed

  19. Type II transmembrane serine proteases as potential targets for cancer therapy

    Science.gov (United States)

    Murray, Andrew S.; Varela, Fausto A.

    2016-01-01

    Carcinogenesis is accompanied by increased protein and activity levels of extracellular cell-surface proteases that are capable of modifying the tumor micro-environment by directly cleaving the extracellular matrix, as well as activating growth factors and proinflammatory mediators involved in proliferation and invasion of cancer cells, and recruitment of inflammatory cells. These complex processes ultimately potentiate neoplastic progression leading to local tumor cell invasion, entry into the vasculature, and metastasis to distal sites. Several members of the type II transmembrane serine protease (TTSP) family have been shown to play critical roles in cancer progression. In this review the knowledge collected over the past two decades about the molecular mechanisms underlying the pro-cancerous properties of selected TTSPs will be summarized. Furthermore, we will discuss how these insights may facilitate the translation into clinical settings in the future by specifically targeting TTSPs as part of novel cancer treatment regimens. PMID:27078673

  20. Cloning and analysis of a Trichinella pseudospiralis muscle larva secreted serine protease gene.

    Science.gov (United States)

    Cwiklinski, Krystyna; Meskill, Diana; Robinson, Mark W; Pozio, Eduardo; Appleton, Judith A; Connolly, Bernadette

    2009-02-23

    Nematode parasites of the genus Trichinella are intracellular and distinct life cycle stages invade intestinal epithelial and skeletal muscle cells. Within the genus, Trichinella spiralis and Trichinella pseudospiralis exhibit species-specific differences with respect to host-parasite complex formation and host immune modulation. Parasite excretory-secretory (ES) proteins play important roles at the host-parasite interface and are thought to underpin these differences in biology. Serine proteases are among the most abundant group of T. spiralis ES proteins and multiple isoforms of the muscle larvae-specific TspSP-1 serine protease have been identified. Recently, a similar protein (TppSP-1) in T. pseudospiralis muscle larvae was identified. Here we report the cloning and characterisation of the full-length transcript of TppSP-1 and present comparative data between TspSP-1 and TppSP-1. PMID:19054614

  1. Small Molecule-Induced Allosteric Activation of the Vibrio Cholerae RTX Cysteine Protease Domain

    Energy Technology Data Exchange (ETDEWEB)

    Lupardus, P.J.; Shen, A.; Bogyo, M.; Garcia, K.C.

    2009-05-19

    Vibrio cholerae RTX (repeats in toxin) is an actin-disrupting toxin that is autoprocessed by an internal cysteine protease domain (CPD). The RTX CPD is efficiently activated by the eukaryote-specific small molecule inositol hexakisphosphate (InsP{sub 6}), and we present the 2.1 angstrom structure of the RTX CPD in complex with InsP{sub 6}. InsP{sub 6} binds to a conserved basic cleft that is distant from the protease active site. Biochemical and kinetic analyses of CPD mutants indicate that InsP{sub 6} binding induces an allosteric switch that leads to the autoprocessing and intracellular release of toxin-effector domains.

  2. Characterizing proteases in an Antarctic Janthinobacterium sp. isolate:Evidence of a protease horizontal gene transfer event

    Institute of Scientific and Technical Information of China (English)

    Cecilia Martinez-Rosales; Juan Jos Marizcurrena; Andrs Iriarte; Natalia Fullana; Hctor Musto; Susana Castro-Sowinski

    2015-01-01

    We report the isolation of a cold-adapted bacterium belonging to the genus Janthinobacterium (named AU11), from a water sample collected in Lake Uruguay (King George Island, South Shetlands). AU11 (growth between 4°C and 30°C) produces a single cold-active extracellular protease (ExPAU11), differentially expressed at low temperature. ExPAU11 was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) as an alkaline metallo-protease (70% coverage with an extracellular protease of Janthinobacterium sp. PI12), and by protease-inhibitor screening identified as a serine-protease. To the best of our knowledge this is the first experimental evidence of a cold-active extracellular protease produced by Janthinobacterium. Furthermore, we identified a serine-protease gene (named JSP8A) showing 60% identity (98%query coverage) to subtilisin peptidases belonging to the S8 family (S8A subfamily) of many cyanobacteria. A phylogenetic analysis of the JSP8A protease, along with related bacterial protein sequences, confirms that JSP8A clusters with S8A subtilisin sequences from different cyanobacteria, and is clearly separated from S8A bacterial sequences of other phyla (including its own). An analysis of the genomic organization around JSP8A suggests that this protease gene was acquired in an event that duplicated a racemase gene involved in transforming L- to D-amino acids. Our results suggest that AU11 probably acquired this subtilisin-like protease gene by horizontal gene transfer (HGT) from a cyanobacterium. We discuss the relevance of a bacterial protease-HGT in the Antarctic environment in light of this hypothesis.

  3. Control of exocellular proteases in dermatophytes and especially Trichophyton rubrum.

    Science.gov (United States)

    Meevootisom, V; Niederpruem, D J

    1979-06-01

    The production of proteases was investigated during growth of dermatophytic fungi with special emphasis on Trichophyton rubrum. Exogenous glucose suppressed elastase production in all dermatophytes examined. The production of protease active guinea pig hair in keratin-salts broth by Microsporum gypseum. Trichophyton mentagrophytes and T. rubrum was also suppressed by glucose. Various carbohydrates added to keratin-salts broth curtailed protease production by T. rubrum as did individual amino acids but ammonium phosphate did not. Enzyme activities against guinea pig hair were compared in twenty-one diverse clinical isolates of T. rubrum cultured in keratin-salts broth. Activity also occurred towards casein, bovine serum albumin, keratin, collagen and elastin after keratin-growth. Studies concerning the properties of enzyme activities in culture filtrates of T. rubrum after keratin-growth suggested that multiple proteases occurred here. Hydrolysis of guinea pig hair and elastin were optimal at pH7 while keratinase was most active at alkaline pH. Divalent cations stimulated protease(s). Ferric ion and mercuric ion stimulated keratinase but were inhibitory to guinea pig hair hydrolysis and elastase. Chelating agents inhibited elastase and the hydrolysis of guinea pig hair more severely than keratinase and all of those effects were reversed by excess calcium. A serine-protease inhibitor, phenylmethylsulfonylfluoride (PMSF), curtailed keratinase but was less inhibitory to elastase and guinea pig hair hydrolysis. Soybean trypsin inhibitor arrested each protease.

  4. Isolation and characterization of proteases from Bacteroides melaninogenicus.

    OpenAIRE

    Fujimura, S.; Nakamura, T.(International Center for Elementary Particle Physics and Department of Physics, The University of Tokyo, Tokyo, Japan)

    1981-01-01

    We isolated two types of intracellular proteases from a strain of Bacteroides melaninogenicus. These enzymes were extracted from cells by ultrasonic treatment and were partially purified. These two enzymes (proteases I and II) differed in molecular weight, heat stability, sensitivity to reducing agents, Km value, and optimum pH for activity.

  5. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Science.gov (United States)

    TECHNICAL ABSTRACT 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 the enzyme had...

  6. Peptide synthesis in neat organic solvents with novel thermostable proteases

    NARCIS (Netherlands)

    Toplak, Ana; Nuijens, Timo; Quaedflieg, Peter J L M; Wu, Bian; Janssen, Dick B

    2015-01-01

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the

  7. Immunoglobulin A1 protease activity in Gemella haemolysans

    DEFF Research Database (Denmark)

    Lomholt, JA; Kilian, Mogens

    2000-01-01

    that cleaves the Pro(227)-Thr(228) peptide bond in the hinge region of the alpha1 chain like that of several Streptococcus species. Phenotypic characterization of the isolates demonstrates that screening for IgA1 protease activity provides a valuable means for species differentiation in this group of bacteria.......The purpose of this study was to determine the occurrence and nature of immunoglobulin A1 (IgA1) protease activity in members of the genus Gemella and related taxa. Among a total of 22 Gemella strains belonging to the four species Gemella haemolysans, Gemella morbillorum, Gemella sanguinis......, and Gemella bergeriae and four reference strains of the species Helcococcus kunzii, Facklamia hominis, and Globicatella sanguinis, IgA1 protease activity was an exclusive character of all nine isolates of G. haemolysans. The IgA1 protease of G. haemolysans appears to be a metallo-type IgA1 protease...

  8. The maize cystatin CC9 interacts with apoplastic cysteine proteases.

    Science.gov (United States)

    van der Linde, Karina; Mueller, André N; Hemetsberger, Christoph; Kashani, Farnusch; van der Hoorn, Renier A L; Doehlemann, Gunther

    2012-11-01

    In a recent study we identified corn cystain9 (CC9) as a novel compatibility factor for the interaction of the biotrophic smut fungus Ustilago maydis with its host plant maize. CC9 is transcriptionally induced during the compatible interaction with U. maydis and localizes in the maize apoplast where it inhibits apoplastic papain-like cysteine proteases. The proteases are activated during incompatible interaction and salicylic acid (SA) treatment and, in turn, are sufficient to induce SA signaling including PR-gene expression. Therefore the inhibition of apoplastic papain-like cysteine proteases by CC9 is essential to suppress host immunity during U. maydis infection. Here were present new experimental data on the cysteine protease-cystatin interaction and provide an in silco analysis of plant cystatins and the identified apoplastic cysteine proteases.

  9. Purification and Characterization of An Alkaline Protease from Acetes chinensis

    Institute of Scientific and Technical Information of China (English)

    XU Jiachao; LIU Xin; LI Zhaojie; XU Jie; XUE Changhu; GAO Xin

    2005-01-01

    An alkaline protease from Acetes chinensis was purified and characterized in this study. The steps of purification include ammonium sulfate precipitation, ion-exchange chromatography with Q-sepharose Fast Flow, gel filtration chromatography with S300 and the second ion-exchange chromatography with Q-sepharose Fast Flow. The protease was isolated and purified, which was present and active on protein substrates (azocasein and casein). The specific protease activity was 17.15folds and the recovery was 4.67. The molecular weight of the protease was estimated at 23.2 kD by SDS-PAGE. With azocasein as the susbstrate, the optimal temperature was 55 ℃ and the optimal pH value was 5.5. Ion Ca2+ could enhance the proteolytic activity of the protease, while Cu2+ , EDTA and PMSF could inhibit its activity.

  10. 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...... 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-protease...... discovered that the mupain-1 scaffold is highly versatile, based on which mupain-1 is potentially able to be retargeted to other serine proteases in the trypsin-like clan. With the scaffold of mupain-1, we rationally designed three inhibitors with high affinity and specificity for another serine protease...

  11. Alkaline Protease Production by a Strain of Marine Yeasts

    Institute of Scientific and Technical Information of China (English)

    WANG Ping; CHI Zhenming; MA Chunling

    2006-01-01

    Yeast strain 10 with high yield of protease was isolated from sediments of saltern near Qingdao, China.The protease had the highest activity at pH 9.0 and 45 ℃.The optimal medium for the maximum alkaline protease production of strain 10 was 2.5 g soluble starch and 2.0 g NaNO3 in 100 mL seawater with initial pH6.0.The optimal cultivation conditions for the maximum protease production were temperature 24.5 ℃, aeration rate 8.0 L min -1 and agitation speed 150 r min-1.Under the optimal conditions, 623.1 Umg-1 protein of alkaline protease was reached in the culture within 30 h of fermentation.

  12. Identification of a senescence-related protease in coriander leaves

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    Senescence-related protease may play an important role in leaf senescence. By improved SDS-Gela- tin-PAGE assay, a 63 ku senescence-related protease (63 SRP) in coriander leaves was identified. Activity of 63 SRP was increased in parallel to the advance of coriander leaf senescence, and inhibited by treating the leaf with gibberellic acid, and enhanced by ethylene treatment. The 63 SRP was suggested to be a serine protease based on the fact that its activity was inhibited by the protease inhibitor PMSF. The optimal temperature for the activity of the 70 ku protease was 50℃. The maximal activity was observed at pH 6-9, some activity could be observed on the gel slices incubated at pH 5 or 11. The 63 SRP was partly purified by the way of ammonium sulfate precipitation and then gel slicing after gel electrophoresis.

  13. FMDV-induced stress granules are disrupted by the viral L-protease

    DEFF Research Database (Denmark)

    Polacek, Charlotta; Belsham, Graham; McInerney, Gerald

    2014-01-01

    have found that foot-and-mouth disease virus (FMDV) triggers SG formation early during infection in IBRS-2 cells. These SGs contain G3BP and TIA-1, but not dsRNA. However, the presence of the FMDV-induced SGs is transient due to the cleavage of G3BP by the viral L-protease (Lpro), which results...... in subsequent SG dispersal. Cells infected with an Lpro-deficient mutant FMDV are not subjected to G3BP cleavage and the SGs formed upon infection with this mutant maintain throughout the infection. In vitro studies using different variants of the Lpro show different G3BP cleavage efficiencies, suggesting...... a superior function of the full length Lpro for this substrate. Furthermore, the Lpro-directed G3BP cleavage is not dependent on virus replication, as investigated by transfecting FMDV RNAs lacking a functional 3D-polymerase. Finally, FMDV RNAs that contain Lpro, but lack the FMDV 3C-protease, also induce...

  14. An outburst scenario for the X-ray spectral variability in 3C 111

    CERN Document Server

    Tombesi, F; Reynolds, C S; Garcia, J; Lohfink, A

    2013-01-01

    We present a combined Suzaku and Swift BAT broad-band E=0.6-200keV spectral analysis of three 3C 111 observations obtained in 2010. The data are well described with an absorbed power-law continuum and a weak (R~0.2) cold reflection component from distant material. We constrain the continuum cutoff at E_c~150-200keV, which is in accordance with X-ray Comptonization corona models and supports claims that the jet emission is only dominant at much higher energies. Fe XXVI Ly\\alpha emission and absorption lines are also present in the first and second observations, respectively. The modelling and interpretation of the emission line is complex and we explore three possibilities. If originating from ionized disc reflection, this should be emitted at r_in> 50r_g or, in the lamp-post configuration, the illuminating source should be at a height of h> 30r_g over the black hole. Alternatively, the line could be modeled with a hot collisionally ionized plasma with temperature kT = 22.0^{+6.1}_{-3.2} keV or a photo-ionized...

  15. Neuronal expression of ILEI/FAM3C and its reduction in Alzheimer's disease.

    Science.gov (United States)

    Liu, Lei; Watanabe, Naoki; Akatsu, Hiroyasu; Nishimura, Masaki

    2016-08-25

    Decrease in brain amyloid-β (Aβ) accumulation is a leading strategy for treating Alzheimer's disease (AD). However, the intrinsic mechanism of the regulation of brain Aβ production is largely unknown. Previously, we reported that ILEI (also referred to as FAM3C) binds to the γ-secretase complex and suppresses Aβ production without inhibiting γ-secretase activity. In this study, we examined ILEI expression in mouse brain using immunohistochemistry and subcellular fractionation. Brain ILEI showed widespread expression in neurons and ependymal cells but not in glial and vascular endothelial cells. Neuronal ILEI resided in perinuclear vesicular structures, which were positive for a marker protein of the trans-Golgi network. Although ILEI immunostaining was negative at synaptic terminals, synaptosome fractionation analysis suggested that ILEI was enriched in presynaptic terminals, particularly in the active zone-docked synaptic vesicles. ILEI expression levels in brain peaked during the postnatal period and declined with age. In comparison with age-matched control brains, the number of ILEI-immunoreactive neurons decreased in AD brains, although the subcellular localization was unaltered. Our results suggest that a decline of ILEI expression may cause accumulation of Aβ in the brain and the eventual development of AD. PMID:27256505

  16. The Central Engine Structure of 3C120: Evidence for a Retrograde Black Hole or a Refilling Accretion Disk

    OpenAIRE

    Cowperthwaite, Philip S.; Reynolds, Christopher S.

    2012-01-01

    The broad-line radio galaxy 3C120 is a powerful source of both X-ray and radio emission including superluminal jet outflows. We report on our reanalysis of 160 ks of Suzaku data taken in 2006, previously examined by Kataoka et al. (2007). Spectral fits to the XIS and HXD/PIN data over a range of 0.7-45 keV reveal a well-defined iron K line complex with a narrow Ka core and relativistically broadened features consistent with emission from the inner regions of the accretion disk. Furthermore, t...

  17. Effect of psychosocial stress on FKBP5 and NR3C1 gene expression in healthy young men

    OpenAIRE

    Nina Höhne; Hildegard Pfister; Tanja Brückl; Petra Zimmermann; Manfred Uhr; Florian Holsboer; Marcus Ising

    2012-01-01

    Stress diseases such as affective disorders are often characterized by a disturbed regulation of the hypothalamus-pituitary-adrenocortical (HPA) axis. This dysregulation can be explained by an impaired function of the receptors involved in the HPA-axis regulation, for example, the glucocorticoid receptors (GR). The regulation process of the HPA axis and the GR function are influenced by several genes, for instance by NR3C1 coding for the GR and also by FKBP5, a co-chaperone in the GR-complex....

  18. Investigation of the angular structure of the quasar 3C196 radio emission

    International Nuclear Information System (INIS)

    The angular dimensions and spectral characteristics are determined for the components of the preferrable brightness distribution model of the quasar 3C196 at decameter and shorter wavelengths. The physical processes are discussed that might be responsible for the extended region of radio emission that has been detected in 3C196 at decameter wavelengths

  19. 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) constit......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......) constitutes an excellent adsorptive method for efficient extracellular protease removal during cultivation. In this work, the impact of semi‐continuous ISMS on the overall protease yield has been investigated. Results reveal significant removal of the protease from Bacillus licheniformis cultivations....... Bacitracin‐functionalized magnetic particles were successfully applied, regenerated and reused up to 30 times. Immediate reproduction of the protease after ISMS proved the biocompatibility of this integrated approach. Six subsequent ISMS steps significantly increased the overall protease yield up to 98...

  20. Molecular Mechanisms of Viral and Host Cell Substrate Recognition by Hepatitis C Virus NS3/4A Protease

    Energy Technology Data Exchange (ETDEWEB)

    Romano, Keith P.; Laine, Jennifer M.; Deveau, Laura M.; Cao, Hong; Massi, Francesca; Schiffer, Celia A. (UMASS, MED)

    2011-08-16

    Hepatitis C NS3/4A protease is a prime therapeutic target that is responsible for cleaving the viral polyprotein at junctions 3-4A, 4A4B, 4B5A, and 5A5B and two host cell adaptor proteins of the innate immune response, TRIF and MAVS. In this study, NS3/4A crystal structures of both host cell cleavage sites were determined and compared to the crystal structures of viral substrates. Two distinct protease conformations were observed and correlated with substrate specificity: (i) 3-4A, 4A4B, 5A5B, and MAVS, which are processed more efficiently by the protease, form extensive electrostatic networks when in complex with the protease, and (ii) TRIF and 4B5A, which contain polyproline motifs in their full-length sequences, do not form electrostatic networks in their crystal complexes. These findings provide mechanistic insights into NS3/4A substrate recognition, which may assist in a more rational approach to inhibitor design in the face of the rapid acquisition of resistance.

  1. PEGylated substrates of NSP4 protease: A tool to study protease specificity

    Science.gov (United States)

    Wysocka, Magdalena; Gruba, Natalia; Grzywa, Renata; Giełdoń, Artur; Bąchor, Remigiusz; Brzozowski, Krzysztof; Sieńczyk, Marcin; Dieter, Jenne; Szewczuk, Zbigniew; Rolka, Krzysztof; Lesner, Adam

    2016-03-01

    Herein we present the synthesis of a novel type of peptidomimetics composed of repeating diaminopropionic acid residues modified with structurally diverse heterobifunctional polyethylene glycol chains (abbreviated as DAPEG). Based on the developed compounds, a library of fluorogenic substrates was synthesized. Further library deconvolution towards human neutrophil serine protease 4 (NSP4) yielded highly sensitive and selective internally quenched peptidomimetic substrates. In silico analysis of the obtained peptidomimetics revealed the presence of an interaction network with distant subsites located on the enzyme surface.

  2. Dual Pressure from Antiretroviral Therapy and Cell-Mediated Immune Response on the Human Immunodeficiency Virus Type 1 Protease Gene

    Science.gov (United States)

    Karlsson, Annika C.; Deeks, Steven G.; Barbour, Jason D.; Heiken, Brandon D.; Younger, Sophie R.; Hoh, Rebecca; Lane, Meghan; Sällberg, Matti; Ortiz, Gabriel M.; Demarest, James F.; Liegler, Teri; Grant, Robert M.; Martin, Jeffrey N.; Nixon, Douglas F.

    2003-01-01

    Human immunodeficiency virus (HIV)-specific CD8+ T-lymphocyte pressure can lead to the development of viral escape mutants, with consequent loss of immune control. Antiretroviral drugs also exert selection pressures on HIV, leading to the emergence of drug resistance mutations and increased levels of viral replication. We have determined a minimal epitope of HIV protease, amino acids 76 to 84, towards which a CD8+ T-lymphocyte response is directed. This epitope, which is HLA-A2 restricted, includes two amino acids that commonly mutate (V82A and I84V) in the face of protease inhibitor therapy. Among 29 HIV-infected patients who were treated with protease inhibitors and who had developed resistance to these drugs, we show that the wild-type PR82V76-84 epitope is commonly recognized by cytotoxic T lymphocytes (CTL) in HLA-A2-positive patients and that the CTL directed to this epitope are of high avidity. In contrast, the mutant PR82A76-84 epitope is generally not recognized by wild-type-specific CTL, or when recognized it is of low to moderate avidity, suggesting that the protease inhibitor-selected V82A mutation acts both as a CTL and protease inhibitor escape mutant. Paradoxically, the absence of a mutation at position 82 was associated with the presence of a high-avidity CD8+ T-cell response to the wild-type virus sequence. Our results indicate that both HIV type 1-specific CD8+ T cells and antiretroviral drugs provide complex pressures on the same amino acid sequence of the HIV protease gene and, thus, can influence viral sequence evolution. PMID:12767994

  3. Protease-Mediated Maturation of HIV: Inhibitors of Protease and the Maturation Process

    Directory of Open Access Journals (Sweden)

    Catherine S. Adamson

    2012-01-01

    Full Text Available Protease-mediated maturation of HIV-1 virus particles is essential for virus infectivity. Maturation occurs concomitant with immature virus particle release and is mediated by the viral protease (PR, which sequentially cleaves the Gag and Gag-Pol polyproteins into mature protein domains. Maturation triggers a second assembly event that generates a condensed conical capsid core. The capsid core organizes the viral RNA genome and viral proteins to facilitate viral replication in the next round of infection. The fundamental role of proteolytic maturation in the generation of mature infectious particles has made it an attractive target for therapeutic intervention. Development of small molecules that target the PR active site has been highly successful and nine protease inhibitors (PIs have been approved for clinical use. This paper provides an overview of their development and clinical use together with a discussion of problems associated with drug resistance. The second-half of the paper discusses a novel class of antiretroviral drug termed maturation inhibitors, which target cleavage sites in Gag not PR itself. The paper focuses on bevirimat (BVM the first-in-class maturation inhibitor: its mechanism of action and the implications of naturally occurring polymorphisms that confer reduced susceptibility to BVM in phase II clinical trials.

  4. 基于S3C2440 Nand Flash启动模式的研究%Study of Nand Flash Boot Mode Based on S 3C2440

    Institute of Scientific and Technical Information of China (English)

    韩金利

    2013-01-01

    This paper introduced the status and role of Nand Flash controller in embedded system design ,taking Samsung's 32-bit embedded microprocessor S3C2440 and K9F2G08U0B chip as example ,it detailedly described the features and software configuration of Nand Flash controller .Combining the interface circuit design of Nand Flash controller and S 3C2440 processor ,it proposed a method to verify the Nand Flash boot process in Linux environment ,and demonstrated it by experiments .%介绍了Nand Flash控制器在嵌入式系统设计中的地位和作用。以Samsung公司的32位嵌入式微处理器S3C2440和K9F2G08U0B芯片为例,详细说明了 Nand Flash控制器的功能和软件配置方法。结合 Nand Flash控制器与S3C2440处理器的接口电路设计,给出了一种 Linux环境下验证 Nand Flash启动过程的方法,并在实验中得到证明。

  5. Very high-resolution observations of the radio sources NRAO 150, OJ 287, 3C 273, M87, 1633+38, BL Lacertae, and 3C 454. 3

    Energy Technology Data Exchange (ETDEWEB)

    Kellermann, K.I. (National Radio Astronomy Observatory, Green Bank, WV); Shaffer, D.B.; Purcell, G.H.

    1977-02-01

    Very long baseline interferometer observations made at a wavelength of 2 and 2.8 cm with baselines ranging from 54 to 291 million wavelengths show a number of radio sources with only slightly resolved components, even on the longest baselines; the quasars 1633 + 38 and 3C 454.3, the objects OJ 287 and BL Lac, and the nucleus of M87 (Virgo A, 3C 274) all contain components < or approximately 0.4 milli-arcsec. The smallest component observed is in the core of 3C 454.3, which contains about 50% of the total flux density and is < or approximately 0.2 milli-arcsec in diameter. The compact component in the nucleus of M87 is < or approximately 1.5 light-months across, and contains about one-third of the total flux density of the nucleus at 2.8 cm. NRAO 150 and BL Lac are double: the components of NRAO 150 are separated by 0.6 milli-arcsec, while BL Lac has an elongated structure consisting of a large (1.4 milli-arcsec) component separated by 1.25 milli-arcsec from a smaller (0.5 milli-arcsec) variable one. The present data on 3C 273 are consistent with triple models similar to those discussed previously, but with a somewhat greater apparent separation of components.

  6. Cold-adapted proteases as an emerging class of therapeutics.

    Science.gov (United States)

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications. PMID:25135820

  7. Exploring a new serine protease from Cucumis sativus L.

    Science.gov (United States)

    Nafeesa, Zohara; Shivalingu, B R; Vivek, H K; Priya, B S; Swamy, S Nanjunda

    2015-03-01

    Coagulation is an important physiological process in hemostasis which is activated by sequential action of proteases. This study aims to understand the involvement of aqueous fruit extract of Cucumis sativus L. (AqFEC) European burp less variety in blood coagulation cascade. AqFEC hydrolyzed casein in a dose-dependent manner. The presence of protease activity was further confirmed by casein zymography which revealed the possible presence of two high molecular weight protease(s). The proteolytic activity was inhibited only by phenyl methyl sulphonyl fluoride suggesting the presence of serine protease(s). In a dose-dependent manner, AqFEC also hydrolysed Aα and Bβ subunits of fibrinogen, whereas it failed to degrade the γ subunit of fibrinogen even at a concentration as high as 100 μg and incubation time up to 4 h. AqFEC reduced the clotting time of citrated plasma by 87.65%. The protease and fibrinogenolytic activity of AqFEC suggests its possible role in stopping the bleeding and ensuing wound healing process. PMID:25577345

  8. Identification of covalent active site inhibitors of dengue virus protease

    Directory of Open Access Journals (Sweden)

    Koh-Stenta X

    2015-12-01

    Full Text Available Xiaoying Koh-Stenta,1 Joma Joy,1 Si Fang Wang,1 Perlyn Zekui Kwek,1 John Liang Kuan Wee,1 Kah Fei Wan,2 Shovanlal Gayen,1 Angela Shuyi Chen,1 CongBao Kang,1 May Ann Lee,1 Anders Poulsen,1 Subhash G Vasudevan,3 Jeffrey Hill,1 Kassoum Nacro11Experimental Therapeutics Centre, Agency for Science, Technology and Research (A*STAR, Singapore; 2Novartis Institute for Tropical Diseases, Singapore; 3Program in Emerging Infectious Diseases, Duke-NUS Graduate Medical School, SingaporeAbstract: Dengue virus (DENV protease is an attractive target for drug development; however, no compounds have reached clinical development to date. In this study, we utilized a potent West Nile virus protease inhibitor of the pyrazole ester derivative class as a chemical starting point for DENV protease drug development. Compound potency and selectivity for DENV protease were improved through structure-guided small molecule optimization, and protease-inhibitor binding interactions were validated biophysically using nuclear magnetic resonance. Our work strongly suggests that this class of compounds inhibits flavivirus protease through targeted covalent modification of active site serine, contrary to an allosteric binding mechanism as previously described.Keywords: flavivirus protease, small molecule optimization, covalent inhibitor, active site binding, pyrazole ester derivatives

  9. Cold-adapted proteases as an emerging class of therapeutics.

    Science.gov (United States)

    Fornbacke, Marcus; Clarsund, Mats

    2013-06-01

    Proteases have been used in medicine for several decades and are an established and well tolerated class of therapeutic agent. These proteases were sourced from mammals or bacteria that exist or have adapted to moderate temperatures (mesophilic organisms); however, proteases derived from organisms from cold environments-cold-adapted or psychrophilic proteases-generally have high specific activity, low substrate affinity, and high catalytic rates at low and moderate temperatures. Made possible by greater flexibility, psychrophilic enzymes interact with and transform the substrate at lower energy costs. Cold-adapted proteases have been used in a wide range of applications, including industrial functions, textiles, cleaning/hygiene products, molecular biology, environmental bioremediations, consumer food products, cosmetics, and pharmaceutical production. In addition to these applications, they have also shown promise as therapeutic modalities for cosmeceutical applications (by reducing glabellar [frown] lines) and a number of disease conditions, including bacterial infections (by disrupting biofilms to prevent bacterial infection), topical wound management (when used as a debridement agent to remove necrotic tissue and fibrin clots), oral/dental health management (by removing plaque and preventing periodontal disease), and in viral infections (by reducing the infectivity of viruses, such as human rhinovirus 16 and herpes simplex virus). Psychrophilic proteases with greater activity and stability (than the original organism-derived variant) have been developed; this coupled with available manufacturing recombinant production techniques suggests that cold-adapted proteases have a promising future as a distinct therapeutic class with diverse clinical applications.

  10. 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.

  11. Laundry detergent compatibility of the alkaline protease from Bacillus cereus.

    Science.gov (United States)

    Banik, Rathindra Mohan; Prakash, Monika

    2004-01-01

    The endogenous protease activity in various commercially available laundry detergents of international companies was studied. The maximum protease activity was found at 50 degrees C in pH range 10.5-11.0 in all the tested laundry detergents. The endogenous protease activity in the tested detergents retained up to 70% on incubation at 40 degrees C for 1 h, whereas less than 30% activity was only found on incubation at 50 degrees C for 1 h. The alkaline protease from an alkalophilic strain of Bacillus cereus was studied for its compatibility in commercial detergents. The cell free fermented broth from shake flask culture of the organism showed maximum activity at pH 10.5 and 50 degrees C. The protease from B. cereus showed much higher residual activity (more than 80%) on incubation with laundry detergents at 50 degrees C for 1 h or longer. The protease enzyme from B. cereus was found to be superior over the endogenous proteases present in the tested commercial laundry detergents in comparison to the enzyme stability during the washing at higher temperature, e.g., 40-50 degrees C. PMID:15293947

  12. A noncovalent class of papain-like protease/deubiquitinase inhibitors blocks SARS virus replication

    Energy Technology Data Exchange (ETDEWEB)

    Ratia, Kiira; Pegan, Scott; Takayama, Jun; Sleeman, Katrina; Coughlin, Melissa; Baliji, Surendranath; Chaudhuri, Rima; Fu, Wentao; Prabhakar, Bellur S.; Johnson, Michael E.; Baker, Susan C.; Ghosh, Arun K.; Mesecar, Andrew D. (Loyola); (Purdue); (UIC)

    2008-10-27

    We report the discovery and optimization of a potent inhibitor against the papain-like protease (PLpro) from the coronavirus that causes severe acute respiratory syndrome (SARS-CoV). This unique protease is not only responsible for processing the viral polyprotein into its functional units but is also capable of cleaving ubiquitin and ISG15 conjugates and plays a significant role in helping SARS-CoV evade the human immune system. We screened a structurally diverse library of 50,080 compounds for inhibitors of PLpro and discovered a noncovalent lead inhibitor with an IC{sub 50} value of 20 {mu}M, which was improved to 600 nM via synthetic optimization. The resulting compound, GRL0617, inhibited SARS-CoV viral replication in Vero E6 cells with an EC{sub 50} of 15 {mu}M and had no associated cytotoxicity. The X-ray structure of PLpro in complex with GRL0617 indicates that the compound has a unique mode of inhibition whereby it binds within the S4-S3 subsites of the enzyme and induces a loop closure that shuts down catalysis at the active site. These findings provide proof-of-principle that PLpro is a viable target for development of antivirals directed against SARS-CoV, and that potent noncovalent cysteine protease inhibitors can be developed with specificity directed toward pathogenic deubiquitinating enzymes without inhibiting host DUBs.

  13. An FtsH protease is recruited to the mitochondrion of Plasmodium falciparum.

    Directory of Open Access Journals (Sweden)

    Aiman Tanveer

    Full Text Available The two organelles, apicoplast and mitochondrion, of the malaria parasite Plasmodium falciparum have unique morphology in liver and blood stages; they undergo complex branching and looping prior to division and segregation into daughter merozoites. Little is known about the molecular processes and proteins involved in organelle biogenesis in the parasite. We report the identification of an AAA+/FtsH protease homolog (PfFtsH1 that exhibits ATP- and Zn(2+-dependent protease activity. PfFtsH1 undergoes processing, forms oligomeric assemblies, and is associated with the membrane fraction of the parasite cell. Generation of a transfectant parasite line with hemagglutinin-tagged PfFtsH1, and immunofluorescence assay with anti-PfFtsH1 Ab demonstrated that the protein localises to P. falciparum mitochondria. Phylogenetic analysis and the single transmembrane region identifiable in PfFtsH1 suggest that it is an i-AAA like inner mitochondrial membrane protein. Expression of PfFtsH1 in Escherichia coli converted a fraction of bacterial cells into division-defective filamentous forms implying a sequestering effect of the Plasmodium factor on the bacterial homolog, indicative of functional conservation with EcFtsH. These results identify a membrane-associated mitochondrial AAA+/FtsH protease as a candidate regulatory protein for organelle biogenesis in P. falciparum.

  14. The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin.

    Science.gov (United States)

    Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin; Vogel, Lotte K; Kataoka, Hiroaki; Bugge, Thomas H

    2014-08-01

    The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating the intracellular trafficking and activation of matriptase. In this study, we, therefore, used genetically engineered mice to determine the effect of ablation of endogenous HAI-1 and endogenous HAI-2 on endogenous matriptase expression, subcellular localization, and activation in polarized intestinal epithelial cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase was mechanistically linked to accelerated activation and shedding of the protease caused by loss of prostasin regulation by HAI-2. Taken together, these data indicate that HAI-1 regulates the activity of activated matriptase, whereas HAI-2 has an essential role in regulating prostasin-dependent matriptase zymogen activation.

  15. Characterization of the entire cystatin gene family in barley and their target cathepsin L-like cysteine-proteases, partners in the hordein mobilization during seed germination.

    Science.gov (United States)

    Martinez, Manuel; Cambra, Ines; Carrillo, Laura; Diaz-Mendoza, Mercedes; Diaz, Isabel

    2009-11-01

    Plant cystatins are inhibitors of cysteine-proteases of the papain C1A and legumain C13 families. Cystatin data from multiple plant species have suggested that these inhibitors act as defense proteins against pests and pathogens and as regulators of protein turnover. In this study, we characterize the entire cystatin gene family from barley (Hordeum vulgare), which contain 13 nonredundant genes, and identify and characterize their target enzymes, the barley cathepsin L-like proteases. Cystatins and proteases were expressed and purified from Escherichia coli cultures. Each cystatin was found to have different inhibitory capability against barley cysteine-proteases in in vitro inhibitory assays using specific substrates. Real-time reverse transcription-polymerase chain reaction revealed that inhibitors and enzymes present a wide variation in their messenger RNA expression patterns. Their transcripts were mainly detected in developing and germinating seeds, and some of them were also expressed in leaves and roots. Subcellular localization of cystatins and cathepsin L-like proteases fused to green fluorescent protein demonstrated the presence of both protein families throughout the endoplasmic reticulum and the Golgi complex. Proteases and cystatins not only colocalized but also interacted in vivo in the plant cell, as revealed by bimolecular fluorescence complementation. The functional relationship between cystatins and cathepsin L-like proteases was inferred from their common implication as counterparts of mobilization of storage proteins upon barley seed germination. The opposite pattern of transcription expression in gibberellin-treated aleurones presented by inhibitors and enzymes allowed proteases to specifically degrade B, C, and D hordeins stored in the endosperm of barley seeds.

  16. Towards 3C-3D digital holographic fluid velocity vector field measurement—tomographic digital holographic PIV (Tomo-HPIV)

    International Nuclear Information System (INIS)

    Most unsteady and/or turbulent flows of geophysical and engineering interest have a highly three-dimensional (3D) complex topology and their experimental investigation is in pressing need of quantitative velocity measurement methods that are robust and can provide instantaneous 3C-3D velocity field data over a significant volumetric domain of the flow. This paper introduces and demonstrates a new method that uses multiple digital CCD array cameras to record in-line digital holograms of the same volume of seed particles from multiple orientations. This technique uses the same basic equipment as Tomo-PIV minus the camera lenses, it overcomes the depth-of-field problem of digital in-line holography and does not require the complex optical calibration of Tomo-PIV. The digital sensors can be oriented in an optimal manner to overcome the depth-of-field limitation of in-line holograms recorded using digital CCD or CMOS array cameras, resulting in a 3D reconstruction of the seed particles within the volume of interest, which can subsequently be analysed using 3D cross-correlation PIV analysis to yield a 3C-3D velocity field. A demonstration experiment of Tomo-HPIV using uniform translation with nominally 11 µm diameter seed particles shows that the 3D displacement derived from 3D cross-correlation Tomo-HPIV analysis can be measured within 5% of the imposed uniform translation, where the imposed uniform translation has an estimated standard uncertainty of 4.3%. So this paper proposes a multi-camera digital holographic imaging 3C-3D PIV method, which is identified as tomographic digital holographic PIV or Tomo-HPIV

  17. Towards 3C-3D digital holographic fluid velocity vector field measurement—tomographic digital holographic PIV (Tomo-HPIV)

    Science.gov (United States)

    Soria, J.; Atkinson, C.

    2008-07-01

    Most unsteady and/or turbulent flows of geophysical and engineering interest have a highly three-dimensional (3D) complex topology and their experimental investigation is in pressing need of quantitative velocity measurement methods that are robust and can provide instantaneous 3C-3D velocity field data over a significant volumetric domain of the flow. This paper introduces and demonstrates a new method that uses multiple digital CCD array cameras to record in-line digital holograms of the same volume of seed particles from multiple orientations. This technique uses the same basic equipment as Tomo-PIV minus the camera lenses, it overcomes the depth-of-field problem of digital in-line holography and does not require the complex optical calibration of Tomo-PIV. The digital sensors can be oriented in an optimal manner to overcome the depth-of-field limitation of in-line holograms recorded using digital CCD or CMOS array cameras, resulting in a 3D reconstruction of the seed particles within the volume of interest, which can subsequently be analysed using 3D cross-correlation PIV analysis to yield a 3C-3D velocity field. A demonstration experiment of Tomo-HPIV using uniform translation with nominally 11 µm diameter seed particles shows that the 3D displacement derived from 3D cross-correlation Tomo-HPIV analysis can be measured within 5% of the imposed uniform translation, where the imposed uniform translation has an estimated standard uncertainty of 4.3%. So this paper proposes a multi-camera digital holographic imaging 3C-3D PIV method, which is identified as tomographic digital holographic PIV or Tomo-HPIV.

  18. Doping and stability of 3C-SiC: from thinfilm to bulk growth

    DEFF Research Database (Denmark)

    Jokubavicius, V.; Sun, J.; Linnarsson, M. K.;

    Cubic silicon carbide (3C-SiC) could pave the way for development of advanced electronic and optoelectronic devices. It could be an excellent substrate for growth of nitride and epitaxial graphene layers. Boron doped 3C-SiC films could reach up to 60% efficiency and pave the way for a new solar...... cell technology. Nitrogen and boron doped 3C-SiC layers can depict a new infrared LED. Hexagonal SiC is an excellent substrate for heteropeitaxial growth of 3C-SiC due to excellent compatibility in lattice constant and thermal expansion coefficient. However, the growth of 3C-SiC on such substrates...... is still being followed by a number of obstacles like polytype stabilization and high density of double positioning boundaries in the grown material. The polytype stability during epitaxial growth of doped 3C-SiC has not been explored. Consequently, the polytype stability during bulk growth of doped 3C-SiC...

  19. 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

  20. OPTIMIZATION OF PROTEASE PRODUCTION FROM FUNGI ISOLATED FROM SOIL

    Directory of Open Access Journals (Sweden)

    Sonia Sethi

    2015-07-01

    Full Text Available Fungal strains isolated from soil by serial dilution method were screened for alkaline protease production. Isolate Penicillium chrysogenum the most potent producer of alkaline protease was identified. The isolate showed highest activity in the optimized medium at pH 9.0, temperature 35ºC, with 1% soycake and peptone incubated for 7 days. Proteases represent one of the largest groups of industrial enzymes and find application in detergents, leather industry, food industry, pharmaceutical industry and bioremediation processes.

  1. Design of wide-spectrum inhibitors targeting coronavirus main proteases.

    Directory of Open Access Journals (Sweden)

    Haitao Yang

    2005-10-01

    Full Text Available The genus Coronavirus contains about 25 species of coronaviruses (CoVs, which are important pathogens causing highly prevalent diseases and often severe or fatal in humans and animals. No licensed specific drugs are available to prevent their infection. Different host receptors for cellular entry, poorly conserved structural proteins (antigens, and the high mutation and recombination rates of CoVs pose a significant problem in the development of wide-spectrum anti-CoV drugs and vaccines. CoV main proteases (M(pros, which are key enzymes in viral gene expression and replication, were revealed to share a highly conservative substrate-recognition pocket by comparison of four crystal structures and a homology model representing all three genetic clusters of the genus Coronavirus. This conclusion was further supported by enzyme activity assays. Mechanism-based irreversible inhibitors were designed, based on this conserved structural region, and a uniform inhibition mechanism was elucidated from the structures of Mpro-inhibitor complexes from severe acute respiratory syndrome-CoV and porcine transmissible gastroenteritis virus. A structure-assisted optimization program has yielded compounds with fast in vitro inactivation of multiple CoV M(pros, potent antiviral activity, and extremely low cellular toxicity in cell-based assays. Further modification could rapidly lead to the discovery of a single agent with clinical potential against existing and possible future emerging CoV-related diseases.

  2. Antiferromagnetic resonance in the Mott insulator fcc-Cs3C60.

    Science.gov (United States)

    Suzuki, Yuta; Shibasaki, Seiji; Kubozono, Yoshihiro; Kambe, Takashi

    2013-09-11

    The magnetic ground state of the fcc phase of the Mott insulator Cs3C60 was studied using a low-temperature electron spin resonance technique, and antiferromagnetic resonance (AFMR) below 1.57 K was directly observed at ambient pressure. The AFMR modes for the fcc phase of Cs3C60 were investigated using a conventional two-sublattice model with uniaxial anisotropy, and the spin-flop field was determined to be 4.7 kOe at 1.57 K. The static magnetic exchange interactions and anisotropy field for fcc-Cs3C60 were also estimated.

  3. Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

    Science.gov (United States)

    Mulimani, Veerappa H.; Sudheendra, Kulkarni; Giri, Ashok P.

    2002-01-01

    Redgram (Cajanus cajan L.) extracts have been analyzed for the protease inhibitors using a new, sensitive, simple, and rapid method for detection of electrophoretically separated protease inhibitors. The detection involves equilibrating the gel successively in the protease assay buffer and protease solution, rinsing the gel in assay buffer, and…

  4. Dosimetric properties of alpha-Al(2)O(3):C exposed to ionizing and non-ionizing radiation

    Science.gov (United States)

    Colyott, Leslie Edward

    Scope and method of study. The trapping states of Czochralski-grown α-Al2O3:C were studied using a variety of experimental techniques, including thermoluminescence (TL), phototransferred thermoluminescence (PTTL) and optical absorption measurements. The focus was placed upon those states responsible for the dosimetric behavior of the α- Al2O3:C, following exposure to various forms of ionizing and non-ionizing radiation. Findings and conclusions. The most effective wavelengths for PTTL are in the short wavelength visible to UV range. The phototransfer processes are complex and appear to involve both electrons and holes. PTTL data suggest that the fading is due to the optical stimulation of charge from the traps into the delocalized bands. At short wavelengths the phototransfer of charge from deep traps into the dosimetry traps must be considered and, thus, the exact wavelength dependence is governed by the radiation and thermal history of the sample. The dose dependence of the TL peak suggests an overlap of several peaks resulting from an array of closely spaced energy levels. A dosimeter which measures the integrated ultraviolet-B (UVB) exposure in air or in water was developed as an application of the PTTL properties of α- Al2O3:C. This dosimeter exploits the increased phototransfer efficiency of α- Al2O3:C to light in the UVB region of the spectrum to produce a near-linear dynamic range of over three decades of UVB exposure. TL and PTTL signals are analyzed, using an algorithm which assumes that a distribution of trapping levels are responsible for the observed TL signals. The signals are deconvolved into unique distribution signatures, which enable the discrimination between irradiations due to gamma/beta, alpha and neutrons. Experiments involving the high temperature anneal of α-Al2O3:C powder in an oxygen atmosphere suggest a diffusion of oxygen vacancies out of the crystal lattice under these conditions, resulting in a decrease in F- and F+- centers. TL

  5. Bowman-Birk protease inhibitor from Vigna unguiculata seeds enhances the action of bradykinin-related peptides.

    Science.gov (United States)

    da Cunha Morales Álvares, Alice; Schwartz, Elisabeth Ferroni; Amaral, Nathalia Oda; Trindade, Neidiane Rosa; Pedrino, Gustavo Rodrigues; Silva, Luciano Paulino; de Freitas, Sonia Maria

    2014-01-01

    The hydrolysis of bradykinin (Bk) by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI) and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M-1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus) ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ. PMID:25361421

  6. Bowman-Birk Protease Inhibitor from Vigna unguiculata Seeds Enhances the Action of Bradykinin-Related Peptides

    Directory of Open Access Journals (Sweden)

    Alice da Cunha M. Álvares

    2014-10-01

    Full Text Available The hydrolysis of bradykinin (Bk by different classes of proteases in plasma and tissues leads to a decrease in its half-life. Here, Bk actions on smooth muscle and in vivo cardiovascular assays in association with a protease inhibitor, Black eyed-pea trypsin and chymotrypsin inhibitor (BTCI and also under the effect of trypsin and chymotrypsin were evaluated. Two synthetic Bk-related peptides, Bk1 and Bk2, were used to investigate the importance of additional C-terminal amino acid residues on serine protease activity. BTCI forms complexes with Bk and analogues at pH 5.0, 7.4 and 9.0, presenting binding constants ranging from 103 to 104 M−1. Formation of BTCI-Bk complexes is probably driven by hydrophobic forces, coupled with slight conformational changes in BTCI. In vitro assays using guinea pig (Cavia porcellus ileum showed that Bk retains the ability to induce smooth muscle contraction in the presence of BTCI. Moreover, no alteration in the inhibitory activity of BTCI in complex with Bk and analogous was observed. When the BTCI and BTCI-Bk complexes were tested in vivo, a decrease of vascular resistance and consequent hypotension and potentiating renal and aortic vasodilatation induced by Bk and Bk2 infusions was observed. These results indicate that BTCI-Bk complexes may be a reliable strategy to act as a carrier and protective approach for Bk-related peptides against plasma serine proteases cleavage, leading to an increase in their half-life. These findings also indicate that BTCI could remain stable in some tissues to inhibit chymotrypsin or trypsin-like enzymes that cleave and inactivate bradykinin in situ.

  7. An allosteric modulator of HIV-1 protease shows equipotent inhibition of wild-type and drug-resistant proteases.

    Science.gov (United States)

    Ung, Peter M-U; Dunbar, James B; Gestwicki, Jason E; Carlson, Heather A

    2014-08-14

    NMR and MD simulations have demonstrated that the flaps of HIV-1 protease (HIV-1p) adopt a range of conformations that are coupled with its enzymatic activity. Previously, a model was created for an allosteric site located between the flap and the core of HIV-1p, called the Eye site (Biopolymers 2008, 89, 643-652). Here, results from our first study were combined with a ligand-based, lead-hopping method to identify a novel compound (NIT). NIT inhibits HIV-1p, independent of the presence of an active-site inhibitor such as pepstatin A. Assays showed that NIT acts on an allosteric site other than the dimerization interface. MD simulations of the ligand-protein complex show that NIT stably binds in the Eye site and restricts the flaps. That bound state of NIT is consistent with a crystal structure of similar fragments bound in the Eye site (Chem. Biol. Drug Des. 2010, 75, 257-268). Most importantly, NIT is equally potent against wild-type and a multidrug-resistant mutant of HIV-1p, which highlights the promise of allosteric inhibitors circumventing existing clinical resistance. PMID:25062388

  8. Purification and properties of two protease inhibitors from rat skin inhibiting papain and other SH-proteases.

    Science.gov (United States)

    Järvinen, M

    1976-01-01

    Two papain inhibitors, I1 and I2, from rat skin extract were purified by affinity chromatography on KSCN-modified papain-agarose gel and by gel filtration on Sephadex G-100. I1 had a molecular weight of 74 000, a pI of 4.6, and it contained 4% of carbohydrates. I1 inhibited papain, ficin, bromelain, rat skin benzoylarginine-2-naphthylamide hydrolase, and to a minor extent, rat skin cathepsin C and bovine trypsin. Bovine chymotrypsin or rat skin cathepsin D were not inhibited and benzoylarginine-2-naphthylamide hydrolase was inhibited only at alkaline pH. An inhibitor corresponding to I1 was present in various rat tissues and also in serum. A similar inhibitor was present in the skin of cat, rabbit, guinea pig, and man. I2 had a molecular weight of 13 400, a pI of 4.9 and it contained no carbohydrates. I2 inhibited all thiol proteases tested, but not trypsin, chymotrypsin, or rat skin cathepsin D. I2 formed an equimolar complex with papain and benzoylarginine-2-naphthylamide hydrolase. I2 was present in rat skin, muscle, lung, and small intestine, but not in kidney, liver, or serum. A similar inhibitor was found in skin extracts of cat, rabbit, guinea pig, and man.

  9. Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease.

    Science.gov (United States)

    Louis, John M; Roche, Julien

    2016-07-01

    Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism.

  10. Evolution under Drug Pressure Remodels the Folding Free-Energy Landscape of Mature HIV-1 Protease.

    Science.gov (United States)

    Louis, John M; Roche, Julien

    2016-07-01

    Using high-pressure NMR spectroscopy and differential scanning calorimetry, we investigate the folding landscape of the mature HIV-1 protease homodimer. The cooperativity of unfolding was measured in the absence or presence of a symmetric active site inhibitor for the optimized wild type protease (PR), its inactive variant PRD25N, and an extremely multidrug-resistant mutant, PR20. The individual fit of the pressure denaturation profiles gives rise to first order, ∆GNMR, and second order, ∆VNMR (the derivative of ∆GNMR with pressure); apparent thermodynamic parameters for each amide proton considered. Heterogeneity in the apparent ∆VNMR values reflects departure from an ideal cooperative unfolding transition. The narrow to broad distribution of ∆VNMR spanning the extremes from inhibitor-free PR20D25N to PR-DMP323 complex, and distinctively for PRD25N-DMP323 complex, indicated large variations in folding cooperativity. Consistent with this data, the shape of thermal unfolding transitions varies from asymmetric for PR to nearly symmetric for PR20, as dimer-inhibitor ternary complexes. Lack of structural cooperativity was observed between regions located close to the active site, including the hinge and tip of the glycine-rich flaps, and the rest of the protein. These results strongly suggest that inhibitor binding drastically decreases the cooperativity of unfolding by trapping the closed flap conformation in a deep energy minimum. To evade this conformational trap, PR20 evolves exhibiting a smoother folding landscape with nearly an ideal two-state (cooperative) unfolding transition. This study highlights the malleability of retroviral protease folding pathways by illustrating how the selection of mutations under drug pressure remodels the free-energy landscape as a primary mechanism. PMID:27170547

  11. Stability and selectivity of alkaline proteases in hydrophilic solvents

    DEFF Research Database (Denmark)

    Pedersen, Lars Haastrup; Ritthitham, Sinthuwat; Pleissner, Daniel

    2008-01-01

    Hydrophilic, organic solvents can be used as co-solvents with water to produce one phase systems sustaining optimal mass transfer of substrates and products of mixed polarity in biocatalysed processes. At concentrations below 50 % hydrophilic solvents can even have a stabilising effect on alkaline...... proteases, but at higher concentrations and particularly in anhydrous systems most enzymes including alkaline proteases will denature and consequently loose activity [1]. However, partial denaturing and increased structural flexibility due to the interaction between hydrophilic solvents and alkaline...... proteases has been agued as the primary reasons for increasing activity, influencing regio-selectivity and improving the enantio-selectivity of these enzymes [2]. Alkaline proteases have been shown to be active not only on peptides, but on a wide range of renewable resources for synthesis of biologically...

  12. The Place of protease inhibitors in antiretroviral treatment

    Directory of Open Access Journals (Sweden)

    S.B. Tenore

    2009-10-01

    Full Text Available With the introduction of highly active antiretroviral therapy, a number of drugs have been developed. The best choice concerning which antiretroviral analogs to start is always under discussion, especially in the choice between non-nucleoside reverse transcriptase inhibitors-based therapies and ritonavir-boosted protease inhibitors. Both are proven to control viral replication and lead to immunological gain. The choice between a non-nucleoside analog reverse transcriptase inhibitor and a protease inhibitor as a third antiretroviral drug in the therapy should consider factors related to the individual, as well as the inclusion of the best therapy in the patient's daily activities and potential adherence. The protease inhibitor-based therapies showed similar efficacy among the various inhibitors with characteristics concerning the adverse events from each medicine. For the treatment of protease-resistant patients, darunavir and tipranavir showed good efficacy with higher genetic barrier to resistance.

  13. 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

    , 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......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...... is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high...

  14. Staphylococcal Proteases Aid in Evasion of the Human Complement System

    DEFF Research Database (Denmark)

    Jusko, Monika; Potempa, Jan; Kantyka, Tomasz;

    2014-01-01

    Staphylococcus aureus is an opportunistic pathogen that presents severe health care concerns due to the prevalence of multiple antibiotic-resistant strains. New treatment strategies are urgently needed, which requires an understanding of disease causation mechanisms. Complement is one of the first...... lines of defense against bacterial pathogens, and S. aureus expresses several specific complement inhibitors. The effect of extracellular proteases from this bacterium on complement, however, has been the subject of limited investigation, except for a recent report regarding cleavage of the C3 component...... by aureolysin (Aur). We demonstrate here that four major extracellular proteases of S. aureus are potent complement inhibitors. Incubation of human serum with the cysteine proteases staphopain A and staphopain B, the serine protease V8 and the metalloproteinase Aur resulted in a drastic decrease...

  15. Mutation analysis of the LCE3B/LCE3C genes in Psoriasis

    Directory of Open Access Journals (Sweden)

    Soto Javier

    2010-03-01

    Full Text Available Abstract Background An association between a common deletion comprising the late cornified envelope LCE3B and LCE3C genes (LCE3C_LCE3B-del and Psoriasis (Ps has been reported. The expression of these LCE genes was induced after skin barrier disruption and was also strong in psoriatic lesions. The damage to the skin barrier could trigger an epidermal response that includes the expression of genes involved in the formation of skin barrier. Methods We determined the LCE3C_LCE3B-del genotype in 405 Ps patients and 400 healthy controls from a Northern Spain region (Asturias. These patients and controls were also genotyped for the rs4112788 single nucleotide polymorphism, in strong linkage disequilibrium with the LCE3C_B cluster. The LCE3B and LCE3C gene variant was determined in the patients through SSCA, DHPLC, and direct sequencing. Results Allele and genotype frequencies did not differ between patients and controls for the rs4112788 and LCE3C_LCE3B-del polymorphisms. However, del/del homozygotes were significantly higher among patients with chronic plaque type Ps who did not develop arthritis (p = 0.03; OR = 1.4; 95%CI = 1.03-1.92. The analysis of the coding sequence of LCE3B and LCE3C in the patients who had at least one copy of this showed that only one patient has a no previously reported LCE3B variant (R68C. Conclusion Our work suggested that homozygosity for a common LCE3C_LCE3B deletion contributes to the risk of developing chronic plaque type Ps without psoriatic arthritis. Our work confirmed previous reports that described an association of this marker with only skin manifestations, and supported the concept of different genetic risk factors contributing to skin and joint disease.

  16. Target cell APOBEC3C can induce limited G-to-A mutation in HIV-1.

    Directory of Open Access Journals (Sweden)

    Khaoula Bourara

    2007-10-01

    Full Text Available The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G and APOBEC3F (A3F are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif, expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition.

  17. Target Cell APOBEC3C Can Induce Limited G-to-A Mutation in HIV-1

    Science.gov (United States)

    Bourara, Khaoula; Liegler, Teri J; Grant, Robert M

    2007-01-01

    The evolutionary success of primate lentiviruses reflects their high capacity to mutate and adapt to new host species, immune responses within individual hosts, and, in recent years, antiviral drugs. APOBEC3G (A3G) and APOBEC3F (A3F) are host cell DNA-editing enzymes that induce extensive HIV-1 mutation that severely attenuates viral replication. The HIV-1 virion infectivity factor (Vif), expressed in vivo, counteracts the antiviral activity of A3G and A3F by inducing their degradation. Other APOBECs may contribute more to viral diversity by inducing less extensive mutations allowing viral replication to persist. Here we show that in APOBEC3C (A3C)-expressing cells infected with the patient-derived HIV-1 molecular clones 210WW, 210WM, 210MW, and 210MM, and the lab-adapted molecular clone LAI, viral G-to-A mutations were detected in the presence of Vif expression. Mutations occurred primarily in the GA context and were relatively infrequent, thereby allowing for spreading infection. The mutations were absent in cells lacking A3C but were induced after transient expression of A3C in the infected target cell. Inhibiting endogenous A3C by RNA interference in Magi cells prevented the viral mutations. Thus, A3C is necessary and sufficient for G-to-A mutations in some HIV-1 strains. A3C-induced mutations occur at levels that allow replication to persist and may therefore contribute to viral diversity. Developing drugs that inhibit A3C may be a novel strategy for delaying viral escape from immune or antiretroviral inhibition. PMID:17967058

  18. Synthesis and electrochemical performance of Ti3C2Tx with hydrothermal process

    Science.gov (United States)

    Wang, Libo; Zhang, Heng; Wang, Bo; Shen, Changjie; Zhang, Chuanxiang; Hu, Qianku; Zhou, Aiguo; Liu, Baozhong

    2016-09-01

    In this study, a simple hydrothermal method has been developed to prepare Ti3C2Tx from Ti3AlC2 as a high-performance electrode material for supercapacitors. This method is environmentally friendly and has a low level of danger. The morphology and structure of the Ti3C2Tx can be controlled by hydrothermal reaction time, temperature and NH4F amounts. The prepared Ti3C2Tx was characterized by X-ray diffraction, field emission scanning electron microscopy, Raman spectroscopy, X-ray photoelectron spectroscopy and Brunauer-Emmet-Teller. The results show that the prepared Ti3C2Tx is terminated by O, OH, and F groups. The electrochemical properties of the Ti3C2Tx sample exhibit specific capacitance up to 141 Fcm-3 in 3 M KOH aqueous electrolyte, and even after 1000 cycles, no significant degradation of the volumetric capacitance was observed. These results indicate that the Ti3C2Tx material prepared by this hydrothermal method can be used in high performance supercapacitors.

  19. Microstructure and mechanical properties of thermal sprayed nanostructured Cr3C2-Ni20Cr coatings

    Directory of Open Access Journals (Sweden)

    Cecilio Alvares da Cunha

    2008-06-01

    Full Text Available Cr3C2-Ni20Cr coatings have been used for corrosion and wear resistant applications. However, one of the shortcomings of these coatings is its low hardness, and consequent low wear resistance, for long term high temperature applications. Nanostructured coatings of many materials have exhibited higher hardness and strength compared with conventional coatings of the same material. Consequently, nanostructured coatings of other materials, including Cr3C2-Ni20Cr have been attempted to enhance overall performance. In this study the effects of high energy milling parameters on Cr3C2-25(Ni20Cr powder characteristics as well as the microstructure and mechanical properties of nanostructured Cr3C2-25(Ni20Cr coatings formed by high velocity oxygen fuel (HVOF spraying have been evaluated. The average particle size and crystallite size of milled Cr3C2-25(Ni20Cr powders decreased with increase in milling time and this decrease was more pronounced in nitrogen compared to that in hexane. This difference has been attributed to a cushioning effect in the latter medium. The coatings prepared with milled Cr3C2-25(Ni20Cr powders had a more uniform microstructure, were harder and had higher relative fracture toughness compared with coatings prepared with as-received powders.

  20. Amplified detection of protease activity using porous silicon nanostructures

    Science.gov (United States)

    Orosco, Manuel

    This dissertation will focus on harnessing the optical properties of porous silicon to sense protease activity. Electrochemical etching of polished silicon wafers produces porous silicon with unique optical properties such as Fabry-Perot fringes or a dielectric mirror reflecting specific wavelengths. Porous silicon optical transducers are coupled to a biochemical reaction (protease activity) and optically measured in a label-free manner. The first chapter is an introductory chapter discussing the current methods of detecting protease activity. Also discussed is the use of porous silicon for label-free sensing. The second chapter discusses the use of thin protein layers that are spin coated on the surface of a porous silicon film and excluded from the porous matrix based on size. When active proteases are introduced to the protein layer, small peptide fragments are generated, causing a change in refractive index from low to high. This can be used as a tool to monitor protease activity and amplify the signal to the naked eye. To extend on the second chapter, a double layered porous silicon film with the first layer have large pores and the second layer etched below having small pores was used for sensing protease activity. Proteases are adsorbed into the first layer and introduction of whole protein substrate produces small peptide fragments that can enter the second layer (changing the effective optical thickness). The fourth chapter describes a method of using luminescent transducers coupled to protein films. An "on-off" sensor using protein coated luminescent porous silicon was used to detect a decrease in the intensity of luminescence due to degradation of the protein film. An "off-on" sensor involved a fluorescent dye housed in the porous film and capped with a protein coating. The release of the dye is caused by the action of a protease causing an increase in fluorescent intensity from the dye.

  1. Marine-derived fungi as a source of proteases

    Digital Repository Service at National Institute of Oceanography (India)

    Kamat, T.; Rodrigues, C.; Naik, C.G.

    known sources of extra cellular enzymes and proteases from the genus Aspergillus have been studied extensivelyl. Fungi from marine and estuarine environment have been screened forproteas~production 20 • Fungi associated with marine organisms, due.... The growth and protease activity as observed is presented in Table 2. Amongst the fungal isolates obtained from five marine organisms Aspergillus terreu,\\' group was most , dominant followed by Acremonium fusidioides. The soft coral Sinularia kavarattiensis...

  2. Proteomic Substrate Identification for Membrane Proteases in the Brain

    Science.gov (United States)

    Müller, Stephan A.; Scilabra, Simone D.; Lichtenthaler, Stefan F.

    2016-01-01

    Cell-cell communication in the brain is controlled by multiple mechanisms, including proteolysis. Membrane-bound proteases generate signaling molecules from membrane-bound precursor proteins and control the length and function of cell surface membrane proteins. These proteases belong to different families, including members of the “a disintegrin and metalloprotease” (ADAM), the beta-site amyloid precursor protein cleaving enzymes (BACE), membrane-type matrix metalloproteases (MT-MMP) and rhomboids. Some of these proteases, in particular ADAM10 and BACE1 have been shown to be essential not only for the correct development of the mammalian brain, but also for myelination and maintaining neuronal connections in the adult nervous system. Additionally, these proteases are considered as drug targets for brain diseases, including Alzheimer’s disease (AD), schizophrenia and cancer. Despite their biomedical relevance, the molecular functions of these proteases in the brain have not been explored in much detail, as little was known about their substrates. This has changed with the recent development of novel proteomic methods which allow to identify substrates of membrane-bound proteases from cultured cells, primary neurons and other primary brain cells and even in vivo from minute amounts of mouse cerebrospinal fluid (CSF). This review summarizes the recent advances and highlights the strengths of the individual proteomic methods. Finally, using the example of the Alzheimer-related proteases BACE1, ADAM10 and γ-secretase, as well as ADAM17 and signal peptide peptidase like 3 (SPPL3), we illustrate how substrate identification with novel methods is instrumental in elucidating broad physiological functions of these proteases in the brain and other organs.

  3. Purification and characterization of alkaline protease from Lysinibacillus fusiformis

    OpenAIRE

    Suppiah S*; Sendeshkannan K; Prabakaran P; Rajkumar G; Yasothkumar N

    2012-01-01

    A novel alkaline protease producing bacterium was isolated from the gut of an estuarine fish Etroplus suratensis. The strain was identified by sequencing the fragment of their bacterial 16s rRNA and its homology was 97% closest to the Lysinibacillus fusiformis. An extracellular protease from this organism was purified by acetone precipitation, ion exchange chromatography and gel filtration chromatography methods and the specific activity of the purified enzyme was found to be 20.39 U/mg, 169....

  4. S3C2440A芯片及应用%Introduction and application of CMOS chip S3C2440A

    Institute of Scientific and Technical Information of China (English)

    张豪; 杨春燕; 汪筱阳

    2011-01-01

    S3C2440A produced by SAMSUNG Company is a high performance-to-price ratio CPU chip. This chip is designed to provide hand-held devices and general applications with low-power, and high-performance micro-controller solution in small size. The internal structure and pin function of the S3C2440A are introduced, included each module of this chip. The typical application is introduced, and corresponding circuit is given in the meanwhile.%S3C2440A是由三星公司生产的一种性价比很高的CPU芯片,由于该芯片价格低、功耗低和体积小等显著的特点,主要用于手持设备和一般类型应用的设备。介绍了该芯片的内部结构和管脚功能,并分别对其各个模块进行了介绍。同时给出了该芯片的一种典型应用实例,并画出了相应的电路图。

  5. Vlbi observations of a complete sample of radio galaxies; 4, the radio galaxies NGC2484, 3C109 and 3C382

    CERN Document Server

    Giovannini, G; Venturi, T; Lara, L; Marcaide, J M; Rioja, M J; Spangler, S R; Wehrle, A E

    1994-01-01

    Abstract: We present here new VLBI observations of one FR-I radio galaxy (NGC2484) and two Broad Line FR-II radio galaxies (3C109 and 3C382). For 3C109 new VLA maps are also shown. These sources belong to a complete sample of radio galaxies under study for a better knowledge of their structures at parsec resolution. The parsec structure of these 3 objects is very similar: asymmetric emission which we interpret as the core plus a one-sided jet. The parsec scale jet is always on the same side of the main kpc-scale jet. The limit on the jet to counterjet brightness ratio, the ratio of the core radio power to the total radio power and the Synchrotron-self Compton model allow us to derive some constraints on the jet velocity and orientation with respect to the line of sight. From these data and from those published on 2 other sources of our sample, we suggest that parsec scale jets are relativistic in both FR-I and FR-II radio galaxies and that pc scale properties in FR-I and FR-II radio galaxies are very similar ...

  6. Molecular Recognition of Cobalt(III)-ligated Peptides by Serine Proteases: The Role of Electrostatic Effects

    DEFF Research Database (Denmark)

    Bagger, Sven; Wagner, Kim

    1998-01-01

    A series of peptides with a positively charged cobalt(III)-complex group attached to the carboxylate terminal was synthesized. The behaviour of these metallopeptides as acceptor nucleophiles in acyl transfer reactions catalyzed by the three serine proteases bovine pancreatic à-chymotrypsin, porcine...... pancreatic trypsin, and proteinase K from Tritirachium album was examined. The efficiency of the substrates was assessed by kinetic measurement of the partition between aminolysis and hydrolysis. The results are discussed with special reference to coulombic interactions between the metal-ligated substrates...... and charged residues on the enzyme surfaces. The idea of using the metallopeptides in practical enzymatic peptide synthesis is put forward....

  7. Structure determination of Murine Norovirus NS6 proteases with C-terminal extensions designed to probe protease–substrate interactions

    Directory of Open Access Journals (Sweden)

    Humberto Fernandes

    2015-02-01

    Full Text Available Noroviruses are positive-sense single-stranded RNA viruses. They encode an NS6 protease that cleaves a viral polyprotein at specific sites to produce mature viral proteins. In an earlier study we obtained crystals of murine norovirus (MNV NS6 protease in which crystal contacts were mediated by specific insertion of the C-terminus of one protein (which contains residues P5-P1 of the NS6-7 cleavage junction into the peptide binding site of an adjacent molecule, forming an adventitious protease-product complex. We sought to reproduce this crystal form to investigate protease–substrate complexes by extending the C-terminus of NS6 construct to include residues on the C-terminal (P′ side of the cleavage junction. We report the crystallization and crystal structure determination of inactive mutants of murine norovirus NS6 protease with C-terminal extensions of one, two and four residues from the N-terminus of the adjacent NS7 protein (NS6 1′, NS6 2′, NS6 4′. We also determined the structure of a chimeric extended NS6 protease in which the P4-P4′ sequence of the NS6-7 cleavage site was replaced with the corresponding sequence from the NS2-3 cleavage junction (NS6 4′ 2|3.The constructs NS6 1′ and NS6 2′ yielded crystals that diffracted anisotropically. We found that, although the uncorrected data could be phased by molecular replacement, refinement of the structures stalled unless the data were ellipsoidally truncated and corrected with anisotropic B-factors. These corrections significantly improved phasing by molecular replacement and subsequent refinement.The refined structures of all four extended NS6 proteases are very similar in structure to the mature MNV NS6—and in one case reveal additional details of a surface loop. Although the packing arrangement observed showed some similarities to those observed in the adventitious protease-product crystals reported previously, in no case were specific protease–substrate interactions

  8. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization

    Science.gov (United States)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.

    2010-08-01

    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  9. OMP Peptides Activate the DegS Stress-Sensor Protease by a Relief of Inhibition Mechanism

    Energy Technology Data Exchange (ETDEWEB)

    Sohn, Jungsan; Grant, Robert A.; Sauer, Robert T.; MIT

    2010-03-19

    In the E. coli periplasm, C-terminal peptides of misfolded outer-membrane porins (OMPs) bind to the PDZ domains of the trimeric DegS protease, triggering cleavage of a transmembrane regulator and transcriptional activation of stress genes. We show that an active-site DegS mutation partially bypasses the requirement for peptide activation and acts synergistically with mutations that disrupt contacts between the protease and PDZ domains. Biochemical results support an allosteric model, in which these mutations, active-site modification, and peptide/substrate binding act in concert to stabilize proteolytically active DegS. Cocrystal structures of DegS in complex with different OMP peptides reveal activation of the protease domain with varied conformations of the PDZ domain and without specific contacts from the bound OMP peptide. Taken together, these results indicate that the binding of OMP peptides activates proteolysis principally by relieving inhibitory contacts between the PDZ domain and the protease domain of DegS.

  10. Inherent dynamics within the Crimean-Congo Hemorrhagic fever virus protease are localized to the same region as substrate interactions

    Energy Technology Data Exchange (ETDEWEB)

    Eisenmesser, Elan Z.; Capodagli, Glenn; Armstrong, Geoffrey S.; Holliday, Michael; Isern, Nancy G.; Zhang, Fengli; Pegan, Scott D.

    2015-05-01

    Crimean-Congo Hemorrhagic fever virus (CCHFV) is one of several lethal viruses that encodes for a viral ovarian tumor domain (vOTU), which serves to cleave and remove multiple proteins involved in cellular signaling such as ubiquitin (Ub) and interferon stimulated gene produce 15 (ISG15). Such manipulation of the host cell machinery serves to downregulate the host response and, therefore, complete characterization of these proteases is important. While several structures of the CCHFV vOTU protease have been solved, both free and bound to Ub and ISG15, few structural differences have been found and little insight has been gained as to the dynamic plasticity of this protease. Therefore, we have used NMR relaxation experiments to probe the dynamics of CCHV vOTU, both alone and in complex with Ub, thereby discovering a highly dynamic protease that exhibits conformational exchange within the same regions found to engage its Ub substrate. These experiments reveal a structural plasticity around the N-terminal regions of CCHV vOTU, which are unique to vOTUs, and provide a rationale for engaging multiple substrates with the same binding site.

  11. Effect of protease inhibitors on the sense of taste.

    Science.gov (United States)

    Schiffman, S S; Zervakis, J; Heffron, S; Heald, A E

    1999-10-01

    The purpose of this study was to investigate the taste properties of protease inhibitors which are essential components of drug regimes used to treat human immunodeficiency virus (HIV) infection. In this study, the taste properties of four protease inhibitors (indinavir, ritonavir, saquinavir, and nelfinavir) were investigated in unmedicated HIV-infected patients and healthy controls. Three of the four protease inhibitors (indinavir, ritonavir, and saquinavir) were found to be predominantly bitter (with additional qualities of medicinal, metallic, astringent, sour, and burning). Nelfinavir was found to be relatively tasteless. HIV-infected and uninfected control subjects detected protease inhibitors at similar concentrations, but HIV-infected subjects perceived suprathreshold concentrations as more bitter than controls. Detection thresholds ranged from 0.0061 mM for saquinavir in HIV-infected patients to 0.0702 mM for ritonavir in uninfected control subjects. Suprathreshold studies indicated that protease inhibitors modified the taste perception of a variety of other taste compounds. These results are consistent with clinical findings that protease inhibitors produce taste complaints that can impact patient compliance. PMID:10501290

  12. Proteases induce secretion of collagenase and plasminogen activator by fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Werb, Z.; Aggeler, J.

    1978-04-01

    We have observed that treatment of rabbit synovial fibroblasts with proteolytic enzymes can induce secretion of collagenase (EC 3.4.24.7) and plasminogen activator (EC 3.4.21.-). Cells treated for 2 to 24 hr with plasmin, trypsin, chymotrypsin, pancreatic elastase, papain, bromelain, thermolysin, or ..cap alpha..-protease but not with thrombin or neuraminidase secreted detectable amounts of collagenase within 16 to 48 hr. Treatment of fibroblasts with trypsin also induced secretion of plasminogen activator. Proteases initiated secretion of collagenase (up to 20 units per 10/sup 6/ cells per 24 hr) only when treatment produced decreased cell adhesion. Collagenase production did not depend on continued presence of proteolytic activity or on subsequent cell adhesion, spreading, or proliferation. Routine subculturing with crude trypsin also induced collagenase secretion by cells. Secretion of collagenase was prevented and normal spreading was obtained if the trypsinized cells were placed into medium containing fetal calf serum. Soybean trypsin inhibitor, ..cap alpha../sub 1/-antitrypsin, bovine serum albumin, collagen, and fibronectin did not inhibit collagenase production. Although proteases that induced collagenase secretion also removed surface glycoprotein, the kinetics of induction of cell protease secretion were different from those for removal of fibronectin. Physiological inducers of secretion of collagenase and plasminogen activator by cells have not been identified. These results suggest that extracellular proteases in conjunction with plasma proteins may govern protease secretion by cells.

  13. 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.

  14. Structural studies on Helicobacter pylori ATP-dependent protease, FtsH

    International Nuclear Information System (INIS)

    The crystal structures of the Helicobacter pylori FtsH ATPase domain in the nucleotide-free state and complexed with ADP have been determined. The ATP-dependent protease, FtsH, degrades misassembled membrane proteins for quality control like SecY, subunit a of FoF1-ATPase, and YccA, and digests short-lived soluble proteins in order to control their cellular regulation, including σ32, LpxC and λcII. The FtsH protein has an N-terminal transmembrane segment and a large cytosolic region that consists of two domains, an ATPase and a protease domain. To provide a structural basis for the nucleotide-dependent domain motions and a better understanding of substrate translocation, the crystal structures of the Helicobacter pylori (Hp) FtsH ATPase domain in the nucleotide-free state and complexed with ADP, were determined. Two different structures of HpFtsH ATPase were observed, with the nucleotide-free state in an asymmetric unit, and these structures reveal the new forms and show other conformational differences between the nucleotide-free and ADP-bound state compared with previous structures. In particular, one HpFtsH Apo structure has a considerable rotation difference compared with the HpFtsH ADP complex, and this large conformational change reveals that FtsH may have the mechanical force needed for substrate translocation

  15. Structural studies on Helicobacter pylori ATP-dependent protease, FtsH

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Sung Hyun; Kang, Gil Bu; Song, Hye-Eun; Park, Sang Jin; Bea, Man-Ho; Eom, Soo Hyun, E-mail: eom@gist.ac.kr [Department of Life Science, Cell Dynamics Research Center, Gwangju Institute of Science and Technology, Gwangju 500-712 (Korea, Republic of)

    2008-05-01

    The crystal structures of the Helicobacter pylori FtsH ATPase domain in the nucleotide-free state and complexed with ADP have been determined. The ATP-dependent protease, FtsH, degrades misassembled membrane proteins for quality control like SecY, subunit a of FoF1-ATPase, and YccA, and digests short-lived soluble proteins in order to control their cellular regulation, including σ32, LpxC and λcII. The FtsH protein has an N-terminal transmembrane segment and a large cytosolic region that consists of two domains, an ATPase and a protease domain. To provide a structural basis for the nucleotide-dependent domain motions and a better understanding of substrate translocation, the crystal structures of the Helicobacter pylori (Hp) FtsH ATPase domain in the nucleotide-free state and complexed with ADP, were determined. Two different structures of HpFtsH ATPase were observed, with the nucleotide-free state in an asymmetric unit, and these structures reveal the new forms and show other conformational differences between the nucleotide-free and ADP-bound state compared with previous structures. In particular, one HpFtsH Apo structure has a considerable rotation difference compared with the HpFtsH ADP complex, and this large conformational change reveals that FtsH may have the mechanical force needed for substrate translocation.

  16. Signal Peptidase Cleavage at the Flavivirus C-prM Junction: Dependence on the Viral NS2B-3 Protease for Efficient Processing Requires Determinants in C, the Signal Peptide, and prM

    OpenAIRE

    Stocks, C. E.; Lobigs, M

    1998-01-01

    Signal peptidase cleavage at the C-prM junction in the flavivirus structural polyprotein is inefficient in the absence of the cytoplasmic viral protease, which catalyzes cleavage at the COOH terminus of the C protein. The signal peptidase cleavage occurs efficiently in circumstances where the C protein is deleted or if the viral protease complex is present. In this study, we used cDNA of Murray Valley encephalitis virus (MVE) to examine features of the structural polyprotein which allow this ...

  17. Cystatin protease inhibitors and immune functions.

    Science.gov (United States)

    Zavasnik-Bergant, Tina

    2008-05-01

    Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. They are wide spread in all living organisms (mammals, nematodes, arthropods etc.) and are involved in various biological processes where they regulate normal proteolysis and also take part in disease pathology. Many cystatins show changes in expression and/or localization, as well as changes in secretion, following certain stimuli acting on immune cells. In immune cells, cystatins interfere with antigen processing and presentation, phagocytosis, expression of cytokines and nitric oxide and these ways modify the immune response. Further, it has been suggested that cystatin-type molecules secreted from parasites down-modulate the host immune response. Precise understanding of the regulatory roles on proteolytic enzymes of endogenous and exogenous cystatins, such as those from parasites, will provide us with valuable insight into how immune response could be modulated to treat a specific disease. This review covers some specific functions of individual cystatins, with a particular focus on the relevance of cystatins to the immune response.

  18. Protease encoding microbial communities and protease activity of the rhizosphere and bulk soils of two maize lines with different N uptake efficiency.

    OpenAIRE

    Baraniya, Divyashri; Puglisi, Edoardo; Ceccherini, Maria Teresa; Pietramellara, Giacomo; Giagnoni, Laura; Arenella, Mariarita; Nannipieri, Paolo; Renella, Giancarlo

    2016-01-01

    This study was carried out to understand the interplay of plant Nitrogen Utilizing Efficiency (NEU) with protease activtiy and microbial proteolytic community composition in the rhizosphere and bulk soils. Protease activity, diversity and abundance of protease genes (using DGGE and qPCR respectively of two key bacterial protease encoding genes: alkaline metallo-peptidase (apr) and neutral-metallopeptidases (npr) were monitored in both rhizosphere and bulk soils from two maize in-bred lines L0...

  19. Synthesis and Crystal Structure of Chloro(p-toluidine)[4-(p-tolyl)imino-2-pentanone-3-oximato]-palladium( Ⅱ ), PdCl(p-CH3C6H4NH2)(p-CH3C6H4-iai)

    Institute of Scientific and Technical Information of China (English)

    冯云龙; 刘世雄

    2003-01-01

    The title complex chloro(p-toluidine)[4-(p-tolyl)imino-2-pentanone-3-oximato]-palladium( Ⅱ ), PdCl(p-CH3C6H4NH2)(p-CH3C6H4-iai), C19H22ClN3O2Pd, (p-CH3C6H4NH2 and p-CH3C6H4-iai- are p-toluidine and the anion of 4-(p-tolyl)imino-2-pentanone-3-oximato, respectively), crystallizes in the monoclinic system, space group C2/c with a = 26.082(5), b = 13.521(3),c = 12.578(3) (A), β= 112.18(3)°, Mr = 466.25, V= 4108(2) (A)3, Dc = 1.508 g/cm3, F(000) = 1888,μ(MoKα) = 1.051 mm-1, Z = 8, the final R = 0.0368 and wR = 0.0855 for 3243 observed reflections (Ⅰ≥ 2σ(Ⅰ)). The coordination environment around Pd is a distorted PdClN3 square-plane composed of imino- and oximo-nitrogen from the Schiff base ligand of p-CH3C6H4-iai-, and amino-nitrogen from p-toluidine and chlorine atom.

  20. Hydrogel Microwell Arrays Allow the Assessment of Protease-Associated Enhancement of Cancer Cell Aggregation and Survival

    OpenAIRE

    Dietmar W. Hutmacher; Lutolf, Matthias P.; Daniela Loessner; Judith A. Clements; Stefan Kobel

    2013-01-01

    Current routine cell culture techniques are only poorly suited to capture the physiological complexity of tumor microenvironments, wherein tumor cell function is affected by intricate three-dimensional (3D), integrin-dependent cell-cell and cell-extracellular matrix (ECM) interactions. 3D cell cultures allow the investigation of cancer-associated proteases like kallikreins as they degrade ECM proteins and alter integrin signaling, promoting malignant cell behaviors. Here, we employed a hydrog...

  1. Radiosynthesis of 9{sup 9mT}c(CO){sup 3C}linafloxacin Dithiocarbamate and Its Biological Evaluation as a Potential staphylococcus aureus Infection Radiotracer

    Energy Technology Data Exchange (ETDEWEB)

    Shah, Syed Qaiser; Khan, Mohammad Rafiullah; Ali, Syed Mohammad [Univ. of Peshawar/Univ. of Engineering and Technology Peshawar, Peshawar (Pakistan)

    2011-12-15

    Clinafloxacin dithiocarbamate (CNND) was radiolabeled with technetium 99m ({sup 99mT}c) using [{sup 99mT}c(CO){sub 3}(H{sub 2O}){sub 3]}{sup +a}nd assessed for its radiochemical stability in saline and serum, its in vitro binding with methicillin resistant Staphylococcus aureus (MRSA) and biodistribution in female nude mice (FNM) artificially infected with live and heat killed MRSA. In normal saline (NS) the {sup 99mT}c(CO){sub 3c}linafloxacin dithiocarbamate ({sup 99mT}c(CO){sub 3C}NND) showed radiochemical stability with a maximum value of 99.10{+-}0.20% and remained stable up to 4 h (92.65{+-}0.18%). In human serum at 37.deg.C within 16 h of incubation, 14.85% side products as a result of de tagging developed. Incubation with MRSA gave saturated binding with a maximum value of 72.75{+-}1.20%. Almost six fold higher uptake was seen in the infected and normal muscle. The {sup 99mT}c (CO){sub 3C}NND complex showed a normal route of excretion from the body of the FNM model. The higher stability in NS, HS, saturated in vitro binding with a live strain of MRSA and six fold higher uptake in the target organ showed the {sup 99mT}c(CO){sub 3C}NND complex to be a potential MRSA infection radiotracer.

  2. The role of Serine Proteases and Serine Protease Inhibitors in the migration of Gonadotropin-Releasing Hormone neurons

    Directory of Open Access Journals (Sweden)

    Silverman Ann-Judith

    2002-02-01

    Full Text Available Abstract Background Mechanisms regulating neuronal migration during development remain largely undefined. Extracellular matrix cues, target site released factors, and components of the migratory neurons themselves are likely all coordinated in time and space directing neurons to their appropriate locations. We have studied the effects of proteases and their inhibitors on the extracellular matrix and the consequences to the migration of gonadotropin releasing hormone (GnRH neurons in the embryonic chick. Chick GnRH neurons differentiate in the olfactory epithelium, migrate along the olfactory nerve and enter the forebrain. The accessibility of this coherent cell group make it amenable for studying protease/inhibitor roles in migratory processes. Results Affigel blue beads were used to deliver a serine protease inhibitor, protease nexin-1 (PN-1, and a target protease, trypsin, to the olfactory epithelium coincident with initiation of GnRH neuronal migration. PN-1 inhibited neuronal migration while trypsin accelerated their transit into the CNS. Prior to initiation of migration, neither PN-1 nor trypsin altered the timing of neuronal exit. Trypsin did, however, accelerate the timing of neuronal crossing into the nerve-forebrain junction. Conclusions These data support the hypothesis that protease activity modulates neuronal movements across barriers. Moreover, the data suggest, for the first time, that aspects of GnRH neuronal migration may be cell autonomous but modulated by ECM alterations.

  3. PURIFIKASI DAN KARAKTERISASI PROTEASE DARI BAKTERI PATOGEN Pseudomonas aeruginosa [Purification and Characterization of Protease from Pathogenic Bacteria Pseudomonas aeruginosa

    Directory of Open Access Journals (Sweden)

    Ace Baehaki1

    2008-06-01

    Full Text Available In the last decade, concern on protease as medical target for overcoming bacterial diseases and viral diseases has been rapidly increased because of the obvious involvement of this enzyme in the molecular of the diseases. The purpose of this research was to purify and characterize protease from pathogenic bacteria Pseudomonas aeruginosa. The bacteria were grown in media containing triptone 1%, NaCl 1% and Yeast extract 0,5%. Protease of P.aeruginosa was purified using column chromatography with Sephadex G-100 gel. There were three peaks of enzyme protein, which were detected on fractions 14, 17 and 30. The optimum pH of the extracelluler protease from P. aeruginosa was 8. The optimum temperature of P.aeruginosa protease was 300C. Fe3+ (1dan 5 mM was strong activator and Co2+ was strong inhibitor. Study on the effect of metals ion and spesific inhibitors indicated that protease from P. aeruginosa was serin metaloprotease. The apparent moleculer weights, as determined by SDS-PAGE and zymogram technique, 36 kD and 42 kD.

  4. STUDY ON GRANULATION MECHANISMS OF γ+(Fe,Mn)3C EUTECTICS IN AUSTENITE STEEL COMPOSITES

    Institute of Scientific and Technical Information of China (English)

    G.F. Liang; Z.M. Xu; Q.C. Jiang; J.G. Li

    2003-01-01

    Results presented in this paper contribute to investigation of the granulation mechanisms of γ+(Fe, Mn)3C eutectics in the austenite matrix composites (abbreviated EAMC). The specimens corresponding to the nominal composition of eutectic with controlled RE(Ce)-Mg agent modifier additions have been unidirectional solidified with a constant growth rate of 2.18μm/s at a fixed temperature gradient of 800K/cm using vertical Bridgeman method. With the RE-Mg agent modifier, the transition of solid/liquid (S/L) interface from columnar to dendrite (CDT), refinement and developed branching of γ and (Fe, Mn)3C phases in the eutectics, and the transition of growth style from faceted-nonfaceted (F/NF) to nonfaceted-nonfaceted (NF/NF)for γ and (Fe, Mn)3C phases in the eutectic have been observed and investigated theoretically. Those can explain the granulation of γ+(Fe, Mn)3C eutectics in the as cast because the roundness increases with the developed lateral branching of primary austenite dendrites, refinement of eutectics, and NF/NF growth of γ and (Fe,Mn)3 C phases in the eutectic.

  5. Capacitance of Ti3C2Tx MXene in ionic liquid electrolyte

    Science.gov (United States)

    Lin, Zifeng; Barbara, Daffos; Taberna, Pierre-Louis; Van Aken, Katherine L.; Anasori, Babak; Gogotsi, Yury; Simon, Patrice

    2016-09-01

    Ti3C2Tx MXene, a two-dimensional (2D) early transition metal carbide, has shown an extremely high volumetric capacitance in aqueous electrolytes, but in a narrow voltage window (less than 1.23 V). The utilization of MXene materials in ionic liquid electrolytes with a large voltage window has never been addressed. Here, we report the preparation of the Ti3C2Tx MXene ionogel film by vacuum filtration for use as supercapacitor electrodes operating in 1-Ethyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (EMI-TFSI) neat ionic liquid electrolyte. Due to the disordered structure of the Ti3C2Tx hydrogel film and a stable spacing after vacuum drying, achieved through ionic liquid electrolyte immersion of the Ti3C2Tx hydrogel film, the Ti3C2Tx surface became accessible to EMI+ and TFSI- ions. A capacitance of 70 F g-1 together with a large voltage window of 3 V was obtained at a scan rate of 20 mV s-1 in neat EMI-TFSI electrolyte. The electrochemical signature indicates a capacitive behavior even at a high scan rate (500 mV s-1) and a high power performance. This work opens up the possibilities of using MXene materials with various ionic liquid electrolytes.

  6. Subcellular object quantification with Squassh3C and SquasshAnalyst.

    Science.gov (United States)

    Rizk, Aurélien; Mansouri, Maysam; Ballmer-Hofer, Kurt; Berger, Philipp

    2015-11-01

    Quantitative image analysis plays an important role in contemporary biomedical research. Squassh is a method for automatic detection, segmentation, and quantification of subcellular structures and analysis of their colocalization. Here we present the applications Squassh3C and SquasshAnalyst. Squassh3C extends the functionality of Squassh to three fluorescence channels and live-cell movie analysis. SquasshAnalyst is an interactive web interface for the analysis of Squassh3C object data. It provides segmentation image overview and data exploration, figure generation, object and image filtering, and a statistical significance test in an easy-to-use interface. The overall procedure combines the Squassh3C plug-in for the free biological image processing program ImageJ and a web application working in conjunction with the free statistical environment R, and it is compatible with Linux, MacOS X, or Microsoft Windows. Squassh3C and SquasshAnalyst are available for download at www.psi.ch/lbr/SquasshAnalystEN/SquasshAnalyst.zip. PMID:26554508

  7. Sema3C Promotes the Survival and Tumorigenicity of Glioma Stem Cells through Rac1 Activation

    Directory of Open Access Journals (Sweden)

    Jianghong Man

    2014-12-01

    Full Text Available Different cancer cell compartments often communicate through soluble factors to facilitate tumor growth. Glioma stem cells (GSCs are a subset of tumor cells that resist standard therapy to contribute to disease progression. How GSCs employ a distinct secretory program to communicate with and nurture each other over the nonstem tumor cell (NSTC population is not well defined. Here, we show that GSCs preferentially secrete Sema3C and coordinately express PlexinA2/D1 receptors to activate Rac1/nuclear factor (NF-κB signaling in an autocrine/paracrine loop to promote their own survival. Importantly, Sema3C is not expressed in neural progenitor cells (NPCs or NSTCs. Disruption of Sema3C induced apoptosis of GSCs, but not NPCs or NSTCs, and suppressed tumor growth in orthotopic models of glioblastoma. Introduction of activated Rac1 rescued the Sema3C knockdown phenotype in vivo. Our study supports the targeting of Sema3C to break this GSC-specific autocrine/paracrine loop in order to improve glioblastoma treatment, potentially with a high therapeutic index.

  8. The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus

    Energy Technology Data Exchange (ETDEWEB)

    Prasad, Lata; Leduc, Yvonne; Hayakawa, Koto; Delbaere, Louis T.J. (Saskatchewan)

    2008-06-27

    V8 protease, an extracellular protease of Staphylococcus aureus, is related to the pancreatic serine proteases. The enzyme cleaves peptide bonds exclusively on the carbonyl side of aspartate and glutamate residues. Unlike the pancreatic serine proteases, V8 protease possesses no disulfide bridges. This is a major evolutionary difference, as all pancreatic proteases have at least two disulfide bridges. The structure of V8 protease shows structural similarity with several other serine proteases, specifically the epidermolytic toxins A and B from S. aureus and trypsin, in which the conformation of the active site is almost identical. V8 protease is also unique in that the positively charged N-terminus is involved in determining the substrate-specificity of the enzyme.

  9. TEM Method for Identification of Domains in Materials which Undergo a Pm3m3->R3c or R3c Phase Transition

    DEFF Research Database (Denmark)

    Bilde-Sørensen, Jørgen

    1979-01-01

    The c-axis of the domains formed by a Pm3m→R3c or R3¯c phase transition may lie along any of the four 〈111〉 C directions of the cubic phase. It is shown that the orientation of the c-axis can be determined by TEM by examining whether two suitable 1/3{113}C superlattice reflections are allowed...... or forbidden in the trigonal phase. As an example, the method is used to examine domains in LaAlO3...

  10. On the Distance and Age of the Pulsar Wind Nebula 3C58

    CERN Document Server

    Kothes, Roland

    2010-01-01

    There is a growing community of astronomers presenting evidence that the pulsar wind nebula 3C58 is much older than the connection with the historical supernova of A.D 1181 would indicate. Most of the strong evidence against a young age for 3C58 relies heavily on the assumed distance of 3.2 kpc determined with HI absorption measurements. I have revisited this distance determination based on new HI data from the Canadian Galactic Plane Survey and added newly determined distances to objects in the neighbourhood, which are based on direct measurements by trigonometric parallax. This leads to a new more reliable distance estimate of 2 kpc for 3C58 and makes the connection between the pulsar wind nebula and the historical event from A.D. 1181 once again much more compelling.

  11. Sensitization by UV light of α-Al2O3:C polycrystalline detectors

    International Nuclear Information System (INIS)

    This paper describes an increase in sensitivity to gamma and beta radiation on α-Al2O3:C polycrystalline detector, which has been produced by a sol-gel process, following previous exposure to ultraviolet light. The increased sensitivity of the detector as a function of the exposure time and ultraviolet wavelength was studied. Since the main luminescent centers have emission peaks at different wavelengths, selective measurements of thermoluminescent emission intensity were done, in order to investigate the possible conversion of centers as a result of the exposition to ultraviolet light. Experimental results indicate that the nature and parameters of the luminescent centers in α-Al2O3:C sol-gel material can be very different of those in α-Al2O3:C single crystal. (author)

  12. The radio structure of 3C 309.1 determined by multi-baseline interferometry

    International Nuclear Information System (INIS)

    Hybrid maps are presented of the radio structure of the QSO 3C 309.1 at 1666 MHz from observations with the European VLBI network and with radio-linked interferometers at Jodrell Bank. The maps reveal that 3C 309.1 has a triple structure with a dominant compact self-absorbed core, extended in pa 1620 +- 20, and symmetrically disposed on either side of this are two relatively extended outer lobes defining an overall pa of approximately 900. This dramatic change in position angle from milliarcsec to the arcsec scale is also seen in many of the asymmetric, core-dominated (D2) quasars, and thus 3C 309.1 appears to be intermediate in its properties between the symmetric (D1) and the D2 classes. It may in fact be a D1 quasar observed nearly end-on, with a relativistically enhanced nuclear core. (author)

  13. Tunable violet-blue emission from 3 C-SiC nanowires

    Science.gov (United States)

    Zhu, J.; Wu, H.; Chen, H. T.; Wu, X. L.; Xiong, X.

    2009-04-01

    Bulk quantities of straight and curled cubic silicon carbide nanowires (3 C-SiC NWs) are synthesized from the mixture of ZnS, Si, and C powders. The 3 C-SiC NWs are wrapped by amorphous SiO 2 shells with very thin thicknesses of less than 2.0 nm. The deionized water suspension of the as-made NWs shows a photoluminescence (PL) band centered at 548 nm, and a tunable violet-blue photoluminescence is observed as the excitation wavelength increases from 300 to 375 nm after the SiO 2 shell is removed. The PL band at 548 nm relates to the SiO 2 shell. Careful microstructural observation suggests that the tunable PL originates from the quantum confinement effect of 3 C-SiC nanocrystallites with sizes of several nm at the turning of the curled NWs.

  14. Lowest instrumented vertebra selection in Lenke 3C and 6C scoliosis

    DEFF Research Database (Denmark)

    Wang, Yu; Bünger, Cody; Zhang, Yanqun;

    2012-01-01

    PURPOSE: The aim of this study was to investigate whether or not post-op curve behaviour differs due to different choices of lowest instrumented vertebra (LIV) with reference to lumbar apical vertebra (LAV) in Lenke 3C and 6C scoliosis. METHODS: We reviewed all the AIS cases surgically treated...... in our institution from 2002 through 2008. Inclusion criteria were as follows: (1) patients with Lenke 3C or 6C scoliosis who were treated with posterior pedicle screw-only constructs; (2) 2-year radiographic follow-up. All the included patients were categorized into three groups based on the relative...... surgery. No significant differences were found in thoracic or lumbar correction rate, global coronal balance and incidence rate of trunk shift among the three groups. CONCLUSION: In conclusion, in Lenke 3C and 6C scoliosis, post-op lumbar curve behaviour differs due to different choices of LIV...

  15. An outburst scenario for the X-ray spectral variability in 3C 111

    Science.gov (United States)

    Tombesi, F.; Reeves, J. N.; Reynolds, C. S.; García, J.; Lohfink, A.

    2013-09-01

    We present a combined Suzaku and Swift BAT broad-band E = 0.6-200 keV spectral analysis of three 3C 111 observations obtained in 2010. The data are well described with an absorbed power-law continuum and a weak (R ≃ 0.2) cold reflection component from distant material. We constrain the continuum cutoff at EC ≃ 150-200 keV, which is in accordance with X-ray Comptonization corona models and supports claims that the jet emission is only dominant at much higher energies. Fe XXVI Lyα emission and absorption lines are also present in the first and second observations, respectively. The modelling and interpretation of the emission line is complex and we explore three possibilities. If originating from ionized-disc reflection, this should be emitted at rin ≥ 50 rg or, in the lamp-post configuration, the illuminating source should be at a height of h ≥ 30 rg above the black hole. Alternatively, the line could be modelled with a hot collisionally ionized plasma with temperature kT = 22.0^{+6.1}_{-3.2} keV or a photoionized plasma with log ξ = 4.52^{+0.10}_{-0.16} erg s-1 cm and column density NH > 3 × 1023 cm-2. However, the first and second scenarios are less favoured on statistical and physical grounds, respectively. The blueshifted absorption line in the second observation can be modelled as an ultrafast outflow (UFO) with ionization parameter log ξ = 4.47^{+0.76}_{-0.04} erg s-1 cm, column density N_H = (5.3^{+1.8}_{-1.3})× 10^{22} cm-2 and outflow velocity vout = 0.104 ± 0.006c. Interestingly, the parameters of the photoionized emission model remarkably match those of the absorbing UFO, supporting the possibility that the same material could be responsible for both emission and absorption. We suggest an outburst scenario in which an accretion disc wind, initially lying out of the line of sight and observed in emission, then crosses our view to the source and it is observed in absorption as a mildly relativistic UFO.

  16. GMRT Detection of HI 21cm Associated Absorption towards the = 1.2 Red Quasar 3C 190

    Indian Academy of Sciences (India)

    C. H. Ishwara-Chandra; K. S. Dwarakanath; K. R. Anantharamaiah

    2003-03-01

    We report the GMRT detection of associated HI 21 cm-line absorption in the = 1.1946 red quasar 3C 190. Most of the absorption is blue-shifted with respect to the systemic redshift. The absorption, at ∼ 647.7MHz, is broad and complex, spanning a velocity width of ∼ 600 kms-1. Since the core is self-absorbed at this frequency, the absorption is most likely towards the hotspots. Comparison of the radio and deep optical images reveal linear filaments in the optical which overlap with the brighter radio jet towards the south-west.We therefore suggest that most of the HI 21 cm-line absorption could be occurring in the atomic gas shocked by the south-west jet.

  17. A Dramatic Optical Flare and Microvariability in the Blazar 3C 454.3

    Science.gov (United States)

    Balonek, Thomas J.; Boni, Samantha J.; Chapman, Katie J.; Didio, Nicholas A.; Sabyr, Alina; Stahlin, R. William; Weaver, Zachary R.; Zhang, Saiyang

    2016-06-01

    Following up on the report of optical and gamma-ray activity in the blazar 3C 454.3 by Jorstad (ATel# 9150), Lucarelli et al (ATel #9157), and Bulgarelli et al (ATel #9176), we report optical (R) observations which reveal a brightening of over 2 magnitudes in a 10 day interval between 2016 June 10 and 20. The brightness on June 20 (R = 13.7) is 1.2 magnitudes brighter than reported by Jorstad on June 13, and is the brightest 3C 454.3 has been observed in two years.

  18. Model Research On Synthesis Of Al2O3-C Layers By MOCVD

    Directory of Open Access Journals (Sweden)

    Sawka A.

    2015-06-01

    Full Text Available These are model studies whose aim is to obtain information that would allow development of new technology for synthesizing monolayers of Al2O3-C with adjusted microstructure on cemented carbides. The Al2O3-C layer will constitute an intermediate layer on which the outer layer of Al2O3 without carbon is synthesized. The purpose of the intermediate layer is to block the cobalt diffusion to the synthesized outer layer of Al2O3 and to stop the diffusion of air oxygen to the substrate during the synthesis of the outer layer. This layer should be thin, continuous, dense and uniform in thickness.

  19. SATCOM antenna siting study on P-3C aircraft, volume 1

    Science.gov (United States)

    Bensman, D. A.; Marhefka, R. J.

    1991-09-01

    The NEC-BSC (Basic Scattering Code) was used to study the performance of a SATCOM antenna on a P-3C aircraft. After plate cylinder fields are added to version 3.1 of the NEC-BSC, it is shown that the NEC-BSC can be used to accurately predict the performance of a SATCOM antenna system on a P-3C aircraft. The study illustrates that the NEC-BSC gives good results when compared with scale model measurements provided by Boeing and Lockheed.

  20. SO2-tolerant Pt-MoO3/C catalyst for oxygen reduction reaction

    Institute of Scientific and Technical Information of China (English)

    Xiao Liu; Zidong Wei; Hongmin Wang; Siguo Chen; Xueqiang Qi; Huiliang Gao; Yi Hui; Yang Bai; Lin Guo; Wei Ding

    2014-01-01

    A Pt-MoO3/C catalyst, aimed to eliminate the harmful effect of sulfur dioxide (SO2) on the performance of Pt nanoparticles (NPs) for catalysis of oxygen reduction reaction (ORR) in proton exchange membrane fuel cells (PEMFC), is developed and characterized by TEM, XRD and XPS. The results reveal that Pt-MoO3/C catalyst exhibits not only a higher catalytic activity, but also a better SO2 poisoning resistance and a better recovery performance than the commercial Pt/C catalyst does.

  1. Nuclear structure in the quasar 3C 345 and the Seyfert galaxy NGC 1275

    Energy Technology Data Exchange (ETDEWEB)

    Matveenko, L.I.; Pauliny-Toth, I.I.K.; Kostenko, V.I.; Romney, J.D.; Baath, L.B.

    1985-05-01

    The structure of the quasar 3C 345 and of the Seyfert galaxy NGC 1275 has been studied at lambda = 18 cm with a global VLBI network. Within 3C 345 are a nucleus and a compact feature, both measuring roughly-equal 0''.0003, accompanied by a thin jet that weakens and bends with distance from the nucleus. In NGC 1275 the compact central feature observed at shorter lambda is absent at 18 cm, probably because of synchrotron self-absorption as well as absorption in ionized gas surrounding the nucleus.

  2. Nuclear structure in the quasar 3C 345 and the Seyfert galaxy NGC 1275

    International Nuclear Information System (INIS)

    The structure of the quasar 3C 345 and of the Seyfert galaxy NGC 1275 has been studied at lambda = 18 cm with a global VLBI network. Within 3C 345 are a nucleus and a compact feature, both measuring roughly-equal 0''.0003, accompanied by a thin jet that weakens and bends with distance from the nucleus. In NGC 1275 the compact central feature observed at shorter lambda is absent at 18 cm, probably because of synchrotron self-absorption as well as absorption in ionized gas surrounding the nucleus

  3. First 230 GHz VLBI Fringes on 3C 279 using the APEX Telescope

    OpenAIRE

    J. Wagner; Roy, A L; Krichbaum, T. P.; Alef, W.; Bansod, A.; Bertarini, A.; Güsten, R.; Graham, D.; HODGSON, J; Märtens, R.; Menten, K.; Muders, D.; Rottmann, H.; TUCCARI, G.; Weiss, A

    2015-01-01

    We report about a 230 GHz very long baseline interferometry (VLBI) fringe finder observation of blazar 3C 279 with the APEX telescope in Chile, the phased submillimeter array (SMA), and the SMT of the Arizona Radio Observatory (ARO). We installed VLBI equipment and measured the APEX station position to 1 cm accuracy (1 sigma). We then observed 3C 279 on 2012 May 7 in a 5 hour 230 GHz VLBI track with baseline lengths of 2800 M$\\lambda$ to 7200 M$\\lambda$ and a finest fringe spacing of 28.6 mic...

  4. OSL response of Al2O3:C inlight dot detectors to ultraviolet radiation

    International Nuclear Information System (INIS)

    The commercial dosimeters Al2O3:C InLight Dot and the OSL microStar System reader, both developed by Landauer, were utilized in this work for the detection of ultraviolet radiation. The OSL response of Al2O3:C InLight Dots was obtained in relation to the parameters of irradiance and illumination time using an UV artificial source. The results showed an increase of the OSL response and a tendency to saturation about 1.7 W.m-2 of irradiance and 30 min of UV illumination. (author)

  5. Glomerular C3c localization indicates ongoing immune deposit formation and complement activation in experimental glomerulonephritis.

    OpenAIRE

    Schulze, M.; Pruchno, C. J.; Burns, M.; Baker, P. J.; R. J. Johnson; Couser, W G

    1993-01-01

    In antibody-mediated glomerular disease, deposits of C3 (C3b) are common and are degraded by factor I to C3c and C3d. However, the kinetics of C3b degradation in glomerulonephritis have not been defined. To do this, we studied three models of complement-dependent glomerulonephritis with established C3 deposits (passive Heymann nephritis, cationized immunoglobulin G membranous nephropathy, and concanavalin A-anticoncanavalin A glomerulonephritis). C3b deposition was halted by administration of...

  6. Thermal conductivity of A1B3C26 ternary compounds and their solid solutions

    International Nuclear Information System (INIS)

    Thermal conductivity of ternary compounds A1B3C26 and solid solutions CuGaxIn1-xS2, CuGaxIn1-xSe2, CuInS2xSe2(1-x), was studied by the absolute and relative methods in the temperature range of 300-550 K. Interrelation between thermal conductivity and tetragonal distortion of the ternary compounds A1B3C26 was established. It is shown that concentrational dependence of thermal conductivity for the solid solutions has the minimum near equimolar compositions

  7. Stepwise Versus Concerted Mechanisms in General-Base Catalysis by Serine Proteases.

    Science.gov (United States)

    Uritsky, Neta; Shokhen, Michael; Albeck, Amnon

    2016-01-26

    General-base catalysis in serine proteases still poses mechanistic challenges despite decades of research. Whether proton transfer from the catalytic Ser to His and nucleophilic attack on the substrate are concerted or stepwise is still under debate, even for the classical Asp-His-Ser catalytic triad. To address these key catalytic steps, the transformation of the Michaelis complex to tetrahedral complex in the covalent inhibition of two prototype serine proteases was studied: chymotrypsin (with the catalytic triad) inhibition by a peptidyl trifluoromethane and GlpG rhomboid (with Ser-His dyad) inhibition by an isocoumarin derivative. The sampled MD trajectories of averaged pKa  values of catalytic residues were QM calculated by the MD-QM/SCRF(VS) method on molecular clusters simulating the active site. Differences between concerted and stepwise mechanisms are controlled by the dynamically changing pKa  values of the catalytic residues as a function of their progressively reduced water exposure, caused by the incoming ligand.

  8. Predictors of virologic response to ritonavir-boosted protease inhibitors.

    Science.gov (United States)

    Marcelin, Anne-Genevieve; Flandre, Philippe; Peytavin, Gilles; Calvez, Vincent

    2005-01-01

    The primary mechanism of resistance to protease inhibitors involves the stepwise accumulation of mutations that alter and block the substrate binding site of HIV protease. The large degree of cross-resistance among the different protease inhibitors is a source of considerable concern for the management of patients after treatment failure. Although the output of HIV-resistance tests has been based on therapeutically arbitrary criteria, there is now an ongoing move towards correlating test interpretation with virologic outcomes on treatment. This approach is undeniably superior, in principle, for tests intended to guide drug choices. However, the predictive accuracy of a given stratagem that links genotype or phenotype to drug response is strongly influenced by the study design, data capture and the analytical methodology used to derive it. There is no definitively superior methodology for generating a genotype-response association for use in interpreting a resistance test, and the various approaches used to date all have their strengths and weaknesses. Combining the information of therapeutic drug monitoring and resistance tests is likely to be of greatest clinical utility in antiretroviral-experienced patients harboring HIV strains with reduced susceptibility. The combination of pharmacologic and virologic parameters as a predictor of the virologic response has been merged into the parameter known as "inhibitory quotient". This article discusses the potential interest of the use of inhibitory quotients as an approach for enhancing the potency and durability of boosted protease inhibitors against protease inhibitor-resistant viruses. PMID:16425962

  9. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    Science.gov (United States)

    Shamsi, Tooba Naz; Parveen, Romana; Fatima, Sadaf

    2016-10-01

    This review describes Protease Inhibitors (PIs) which target or inhibit proteases, protein digesting enzymes. These proteases play a crucial task in many biological events including digestion, blood coagulation, apoptosis etc. Regardless of their crucial roles, they need to be checked regularly by PIs as their excess may possibly damage host organism. On basis of amino acid composition of PIs where Protease-PI enzymatic reactions occur i.e. serine, cysteine, and aspartic acid, they are classified. Nowadays, various PIs are being worked upon to fight various parasitic or viral diseases including malaria, schistosomiasis, colds, flu', dengue etc. They prevent an ongoing process begun by carcinogen exposure by keeping a check on metastasis. They also possess potential to reduce carcinogen-induced, increased levels of gene amplification to almost normal levels. Some PIs can principally be used for treatment of hypertension and congestive heart failure by blocking conversion of angiotensin I to angiotensin II for example Angiotensin-converting enzyme inhibitors (ACEIs). Also PIs target amyloid β-peptide (Aβ) level in brain which is prime responsible for development of Alzheimer's Disease (AD). Also, PIs inhibit enzymatic activity of HIV-1 Protease Receptor (PR) by preventing cleavage events in Gag and Gag-Pol that result in production of non-virulent virus particles. PMID:26955746

  10. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    Energy Technology Data Exchange (ETDEWEB)

    Mittal, Seema; Cai, Yufeng; Nalam, Madhavi N.L.; Bolon, Daniel N.A.; Schiffer, Celia A. (UMASS, MED)

    2012-09-11

    Human immunodeficiency virus Type-1 (HIV-1) protease is crucial for viral maturation and infectivity. Studies of protease dynamics suggest that the rearrangement of the hydrophobic core is essential for enzyme activity. Many mutations in the hydrophobic core are also associated with drug resistance and may modulate the core flexibility. To test the role of flexibility in protease activity, pairs of cysteines were introduced at the interfaces of flexible regions remote from the active site. Disulfide bond formation was confirmed by crystal structures and by alkylation of free cysteines and mass spectrometry. Oxidized and reduced crystal structures of these variants show the overall structure of the protease is retained. However, cross-linking the cysteines led to drastic loss in enzyme activity, which was regained upon reducing the disulfide cross-links. Molecular dynamics simulations showed that altered dynamics propagated throughout the enzyme from the engineered disulfide. Thus, altered flexibility within the hydrophobic core can modulate HIV-1 protease activity, supporting the hypothesis that drug resistant mutations distal from the active site can alter the balance between substrate turnover and inhibitor binding by modulating enzyme activity.

  11. Mitochondrial cereblon functions as a Lon-type protease

    Science.gov (United States)

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-01-01

    Lon protease plays a major role in the protein quality control system in mammalian cell mitochondria. It is present in the mitochondrial matrix, and degrades oxidized and misfolded proteins, thereby protecting the cell from various extracellular stresses, including oxidative stress. The intellectual disability-associated and thalidomide-binding protein cereblon (CRBN) contains a large, highly conserved Lon domain. However, whether CRBN has Lon protease-like function remains unknown. Here, we determined if CRBN has a protective function against oxidative stress, similar to Lon protease. We report that CRBN partially distributes in mitochondria, suggesting it has a mitochondrial function. To specify the mitochondrial role of CRBN, we mitochondrially expressed CRBN in human neuroblastoma SH-SY5Y cells. The resulting stable SH-SY5Y cell line showed no apparent effect on the mitochondrial functions of fusion, fission, and membrane potential. However, mitochondrially expressed CRBN exhibited protease activity, and was induced by oxidative stress. In addition, stably expressed cells exhibited suppressed neuronal cell death induced by hydrogen peroxide. These results suggest that CRBN functions specifically as a Lon-type protease in mitochondria. PMID:27417535

  12. Plant collagenase: unique collagenolytic activity of cysteine proteases from ginger.

    Science.gov (United States)

    Kim, Misook; Hamilton, Susan E; Guddat, Luke W; Overall, Christopher M

    2007-12-01

    Two cysteine proteases, GP2 and GP3, have been isolated from ginger rhizomes (Zingiber officinale). GP2 is virtually identical to a previously identified ginger protease GPII [K.H. Choi, and R.A. Laursen, Amino-acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale, Eur. J. Biochem. 267 (2000) 1516-1526.], and cleaves native type I collagen at multiple discrete sites, which are in the interior of the triple helical region of this molecule. In reaction with proline-containing peptides GP2 shows preference for Pro in the P2 position, and at least 10-fold higher efficiency of hydrolysis than papain. Comparison of models of GP2 and GP3 with the crystal structure of papain shows that the three enzymes have different S2 pocket structures. The S2 pocket in GP2 and GP3 is half the size of that of papain. GP2 is the only reported plant cysteine protease with a demonstrated ability to hydrolyse native collagen. The results support a role for ginger proteases as an alternative to papain, in commercial applications such as meat tenderization, where collagen is the target substrate. PMID:17920199

  13. Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

    Science.gov (United States)

    Verma, Amit; Ansari, Mohammad W; Anwar, Mohmmad S; Agrawal, Ruchi; Agrawal, Sanjeev

    2014-05-01

    Proteases have found a wide application in the several industrial processes, such as laundry detergents, protein recovery or solubilization, prion degradation, meat tenderizations, and in bating of hides and skins in leather industries. But the main hurdle in industrial application of proteases is their economical production on a large scale. The present investigation aimed to exploit the locally available inexpensive agricultural and household wastes for alkaline protease production using Thermoactinomyces sp. RS1 via solid-state fermentation (SSF) technique. The alkaline enzyme is potentially useful as an additive in commercial detergents to mitigate pollution load due to extensive use of caustic soda-based detergents. Thermoactinomyces sp. RS1 showed good protease production under SSF conditions of 55 °C, pH 9, and 50 % moisture content with potato peels as solid substrate. The presented findings revealed that crude alkaline protease produced by Thermoactinomyces sp. RS1 via SSF is of potential application in silver recovery from used X-ray films. PMID:24122212

  14. Purification and characterization of an extracellular protease from Clonostachys rosea

    Institute of Scientific and Technical Information of China (English)

    LI Jun; HUANG Xiao-wei; ZHANG Ke-qin

    2004-01-01

    @@ An extracellular protease from Clonostachys rosea (syn. Gliocladium roseum) was purified to SDSPAGE homogeneity with 14-fold purification by ultrafiltration、 ammonium sulfate precipetation、hydrophobic interaction chromatography and anion exchange chromatography. The molecular weight of the protease was 32 kDa as estimated by SDS-PAGE. The N-terminal sequence of first 10 amino acids was A-T-Q-S-N-A-P-W-G-L. This enzyme exhibited pH and temperature optima of 9-10 and 60℃, respectively, and was stable over a wide range of pH 4-10 and temperature 4-50 ℃. It did not require Ca2+ for activity and thermal stability. Pre-incubation of enzyme with Zn2+ , Cu2+ , Hg2+,Fe3+ inhibited most of the enzyme activity, but Mn2+ increased enzyme activity up to 38%. It remained stable in the presence of Tween20, H2O2, EDTA. The inhibition profile of the enzymes by PMSF, suggested that this purified protease belongs to the serine protease family. The protease could immobilize nematodes (Panagrellus redivirus) in bioassays and hydrolyzed proteins of the purified cuticle.

  15. Fatores antinutricionais: inibidores de proteases e lectinas Antinutritional factors: protease inhibitors and lectins

    Directory of Open Access Journals (Sweden)

    Mara Reis SILVA

    2000-04-01

    Full Text Available Os fatores antinutricionais presentes em alimentos podem provocar efeitos fisiológicos adversos ou diminuir a biodisponibilidade de nutrientes. A maior questão sobre os riscos à saúde provocados por antinutrientes é o desconhecimento dos níveis de tolerância, do grau de variação do risco individual e da influência de fatores ambientais sobre a capacidade de detoxificação do organismo humano. Dentre os fatores antinutricionais os inibidores de proteases e as lectinas são considerados instáveis ao tratamento térmico. A hipertrofia pancreática causada pelos inibidores de tripsina tem sido relatada em alguns estudos com animais. As alterações da função fisiológica em animais causadas por ação de lectinas no intestino parecem estar relacionadas à especificidade destas substâncias com as células da mucosa intestinal. Os possíveis efeitos adversos dos inibidores de proteases e das lectinas na maioria das vezes são inferidos somente de experimentos com animais de laboratório.The antinutritional factors present in foods can cause adverse physiological effects or decrease the bioavailability of nutrients. Health risk factors associated with antinutrients include: lack of knowledge of the tolerance levels to these compounds in the human organism, little available information on the degree of variation of individual risks and little knowledge with respect to the influence of environmental factors on the detoxification capacity of the human organism. The possible adverse effects of protease inhibitors and lectin on human health are, in most cases, only inferred by way of experiments with laboratory animals. Pancreatic hypertrophy caused by trypsin inhibitors has been shown in some animal experiments. Alterations in physiological functions at the intestinal level shown by animals submitted to lectins containing diets seem to be related to the specificity of these substances with the intestinal mucosa cells. There is no evidence

  16. DEVELOPMENT OF A 400 LEVEL 3C CLAMPED DOWNHOLE SEISMIC RECEIVER ARRAY FOR 3D BOREHOLE SEISMIC IMAGING OF GAS RESERVOIRS

    Energy Technology Data Exchange (ETDEWEB)

    Bjorn N.P. Paulsson

    2002-09-01

    Borehole seismology is the highest resolution geophysical imaging technique available to the oil and gas industry for characterization and monitoring of oil and gas reservoirs. However, the industry's ability to economically do high resolution 3D imaging of deep and complex gas reservoirs using borehole seismology is currently frustrated by the lack of the acquisition technology necessary to record the large volumes of the high frequency, high signal-to-noise-ratio borehole seismic data needed to do 3D imaging. This proposal takes direct aim at this shortcoming. P/GSI is developing a 400 level 3C clamped downhole seismic receiver array for borehole seismic 3D imaging. This array will remove the acquisition barrier to record the necessary volumes of data to do high resolution 3D VSP or 3D cross well seismic imaging. 3D VSP and long range Cross-Well Seismology (CWS) are two of the borehole seismic techniques that will allow the Gas industry to take the next step in their quest for higher resolution images of the gas reservoirs. Today only a fraction of the original Oil or Gas in place is produced when reservoirs are considered depleted. This is primarily due to our lack of understanding of the detailed compartmentalization of the oil and gas reservoirs. The 400 level 3C borehole seismic receiver array will allow for economic use of 3D borehole seismic imaging for reservoir characterization and monitoring. By using 3C surface seismic or 3C borehole seismic sources the 400 level receiver array will furthermore facilitate 9C reservoir imaging. The 9C borehole seismic data will provide P, SH and SV information for imaging of the complex deep gas reservoirs and allow quantitative prediction of the rock and the fluid types. The data quality and the data volumes from a 400 level 3C array will allow us to develop the data processing technology necessary for high resolution reservoir imaging.

  17. Bacillus licheniformis proteases as high value added products from fermentation of wastewater sludge: pre-treatment of sludge to increase the performance of the process.

    Science.gov (United States)

    Drouin, M; Lai, C K; Tyagi, R D; Surampalli, R Y

    2008-01-01

    Wastewater sludge is a complex raw material that can support growth and protease production by Bacillus licheniformis. In this study, sludge was treated by different thermo-alkaline pre-treatment methods and subjected to Bacillus licheniformis fermentation in bench scale fermentors under controlled conditions. Thermo-alkaline treatment was found to be an effective pre-treatment process in order to enhance the proteolytic activity. Among the different pre-treated sludges tested, a mixture of raw and hydrolysed sludge caused an increase of 15% in the protease activity, as compared to the untreated sludge. The benefit of hydrolysis has been attributed to a better oxygen transfer due to decrease in media viscosity and to an increase in nutrient availability. Foam formation was a major concern during fermentation with hydrolysed sludge. The studies showed that addition of a chemical anti-foaming agent (polypropylene glycol) during fermentation to control foam could negatively influence the protease production by increasing the viscosity of sludge.

  18. Identification of a bacterial-like HslVU protease in the mitochondria of Trypanosoma brucei and its role in mitochondrial DNA replication.

    Directory of Open Access Journals (Sweden)

    Ziyin Li

    2008-04-01

    Full Text Available ATP-dependent protease complexes are present in all living organisms, including the 26S proteasome in eukaryotes, Archaea, and Actinomycetales, and the HslVU protease in eubacteria. The structure of HslVU protease resembles that of the 26S proteasome, and the simultaneous presence of both proteases in one organism was deemed unlikely. However, HslVU homologs have been identified recently in some primordial eukaryotes, though their potential function remains elusive. We characterized the HslVU homolog from Trypanosoma brucei, a eukaryotic protozoan parasite and the causative agent of human sleeping sickness. TbHslVU has ATP-dependent peptidase activity and, like its bacterial counterpart, has essential lysine and N-terminal threonines in the catalytic subunit. By epitope tagging, TbHslVU localizes to mitochondria and is associated with the mitochondrial genome, kinetoplast DNA (kDNA. RNAi of TbHslVU dramatically affects the kDNA by causing over-replication of the minicircle DNA. This leads to defects in kDNA segregation and, subsequently, to continuous network growth to an enormous size. Multiple discrete foci of nicked/gapped minicircles are formed on the periphery of kDNA disc, suggesting a failure in repairing the gaps in the minicircles for kDNA segregation. TbHslVU is a eubacterial protease identified in the mitochondria of a eukaryote. It has a novel function in regulating mitochondrial DNA replication that has never been observed in other organisms.

  19. Screening of Alkaline Protease-Producing Streptomyces diastaticus and Optimization of Enzyme Production

    Directory of Open Access Journals (Sweden)

    Elham Dawoodi

    2014-12-01

    Full Text Available Background and Aim: Alkaline proteases are used in pharmaceutical, film and photography, silk production and food, leather and detergent industries. Actinomycetes are gram positive bacteria that produce different enzymes such as proteases. The aims of this research were isolation of native alkaline protease-producing Actinomycete spp. from different soil samples as well as optimizing the conditions for enzyme production. Materials and Methods: The different soil samples were collected from different locations of the provinces of Khouzestan, Chahar Mahalo Bakhtiari and Isfahan, Iran. After determining of the best alkaline protease producing species using Lowry method, the optimization of alkaline protease was performed. Results: The alkaline protease producing Actinomycete spp. was isolated from soil. The most enzyme activity was measured in S.diastaticus. The best concentration of sucrose as the carbon source for the highest production of alkaline protease was 10 g/l. The optimum pH and temperature for the alkaline protease production by S. diastaticus were 10 and 30°C respectively. The maximum activity of alkaline protease was measured at 200 rpm as the best aeration speed. Conclusions: This is the first report of alkaline protease production by Streptomyces diastaticus in Iran. The accomplished examinations in this research confirmed the previous theories of alkaline protease production by Actinomycetes relatively. Regarding the immense applications of alkaline proteases in several industries and isolation of a native alkaline protease producing Actinomycete, The production potential of this enzyme in our country could be accessible in the near future.

  20. Natural gas C3-C8 Hydrocarbon Enrichment Analysis Methods%天然气C3-C8烃类浓缩分析新方法

    Institute of Scientific and Technical Information of China (English)

    杨晓春; 王璋

    2015-01-01

    天然气形成的原因,但在一般情况下,天然气和油的成因是不同的,在天然气中,所用的有用信息数量不多。根据这一情况,自己配制了以C3—C8烃为主的天然气浓缩器,而当场就可以去井口搜集天然气的、浓缩的样品,在此基础上,形成了以天然气C3—C8烃为主的浓缩方法。对于该方法,能把天然气中的C3—C8烃类物质分析出来,从而完成了任务,即对天然气组分不多、气源相对不易,这是一个新的思路、方法,值得我们学习。%The causes of the formation of natural gas,but in general,the causes of natural gas an d oil are different,in natural gas,the small number of useful information.According to this situation,make myself a predominantly C3 - C8 hydrocarbon gas concentrator,and can go on the wellhead collecting gas,concentration of sample,on this basis,formed mainly gas C3 - C8 hydrocarbon enrichment method.For the method,can change the C3 - C8 hydrocarbons in natural gas analysis,so as to complete the task,namely a few components of natural gas,gas source is relatively difficult,this is a new train of thought,method and worth our learning.

  1. Study of the irradiation defects in 3C-SiC; Etude des defauts d'irradiation dans 3C-SiC

    Energy Technology Data Exchange (ETDEWEB)

    Lefevre, J. [Ecole Polytechnique, 91 - Palaiseau (France). Lab. CEA d' Etudes des Solides Irradies

    2007-07-01

    This work deals with the study of the irradiation defects in the cubic polytype 3C of the n type silicon carbide. Low temperature photoluminescence and electron spin resonance techniques have been used. In situ photoluminescence measurements after irradiation at 10 K by electrons have shown that the nature of the defects induced is identical to those observed after irradiation at ambient temperature with electrons, protons or carbon ions. No regeneration of these defects has been revealed after in situ annealings until 300 K. The electrons Van de Graff accelerator of the Irradiated Solid Laboratory has allowed to irradiate sample of 3C in a range of energies between 190 keV and 1 MeV. It has then been possible to estimate the appearance threshold of the irradiation defects but especially to be able to determine the displacement threshold energy of silicon in this SiC polytype. The found value of 25 eV is in good agreement with the first experimental result proposed by X. Kerbiriou with the use of the ESR. Annealings in the range of high temperatures have been carried out. The evolution of the irradiation defects has been followed in photoluminescence and in ESR. The results show that, in one part, the vacancy of the silicon negatively charged is essentially the only compensating defect in 3C-SiC of n type and that, in another part, the majority of the defects are annealed below 1200 C. Only the D1 defect remains after annealings until 1600 C. The D1 center is in fact a native defect in SiC; indeed, it has been identified alone in non irradiated samples. A systematic study of these last samples show the absence of D1 in samples strongly compensated. The compared results of photoluminescence and of positons annihilation are in good agreement for the possible attribution of D1 to the bi-vacancy V{sub C}-V{sub Si}. One of the most interesting result of this last work has been obtained using the ESR technique under excitation with a neodymium laser. The measurements

  2. 880 $\\mu$m SMA polarization observations of the quasar 3C 286

    CERN Document Server

    Hull, Charles L H; Zhang, Qizhou

    2016-01-01

    For decades the bright radio quasar 3C 286 has been widely recognized as one of the most reliable polarization calibrators at centimeter wavelengths because of its unchanging polarization position angle and high polarization percentage. However, it has become clear in recent years that the polarization position angle of 3C 286 changes with increasing frequency, increasing from ~33$^{\\circ}$ at $\\lambda \\gtrsim 3$ cm to ~38$^{\\circ}$ at $\\lambda \\approx 1$ mm. With the advent of high-sensitivity polarization observations by current and future (sub)millimeter telescopes, knowledge of the position angle of 3C 286 at higher frequencies is critical for calibration. We report the first polarization observations of 3C 286 at submillimeter wavelengths, taken at 880 $\\mu$m (340 GHz) with the Submillimeter Array. We find a polarization position angle and percentage of $37.4 \\pm 1.5^{\\circ}$ and $15.7 \\pm 0.8$%, respectively, consistent with previous measurements at 1 mm.

  3. Thermally stimulated conductivity and thermoluminescence from Al2O3 : C

    DEFF Research Database (Denmark)

    Agersnap Larsen, N.; Bøtter-Jensen, L.; McKeever, S.W.S.

    1999-01-01

    Simultaneous measurements of thermoluminescence (TL) and thermally stimulated conductivity (TSC) are reported on single-crystal dosimetry-quality Al2O3:C following gamma irradiation at room temperature. Analysis of the data reveals a superposition of several first-order TL and TSC peaks caused...

  4. The Angular Structure of Quasar 3C 47 in the Decameter Waveband

    Science.gov (United States)

    Lozinskiy, A. B.; Lozinskiy, R. A.; Ivantishin, O. L.; Romanchev, Y. V.; Rashkovskiy, S. L.; Shepelev, V. A.; Brazhenko, A. I.; Vashchishin, R. V.; Litvinenko, O. A.

    The quasar 3C47 was observed with the URAN network at decameter wavelengths. A model of its angular structure consisting of four components was fitted. Lobes of the source are enlarged significantly in the range if compare with their high frequency dimensions. The hot spots emission is detected at low frequencies but a radio core is disappeared completely due to its flat spectrum.

  5. The structure of X-ray jet of quasar 3C 273

    International Nuclear Information System (INIS)

    The X-ray internal structure of extragalactic jets is analyzed. The Lucy-Richardson deconvolution algorithm is used to restore the image of X-ray sources. The analysis was done for Chandra observations of core-dominated quasar 3C 273. The transverse profiles are built for the jet knots

  6. The Structure & Linear Polarization of the Kiloparsec-Scale Jet of the Quasar 3C\\,345

    CERN Document Server

    Roberts, David H; Marchenko, Valerie V

    2012-01-01

    Deep Very Large Array imaging of the quasar 3C\\,345 at 4.86 and 8.44 GHz has been used to study the structure and linear polarization of its radio jet on scales ranging from 2 to 30 kpc. There is a 7--8 Jy unresolved core with spectral index $\\alpha \\simeq -0.24$ ($I_\

  7. Ti3C2Tx Filler Effect on the Proton Conduction Property of Polymer Electrolyte Membrane.

    Science.gov (United States)

    Liu, Yahua; Zhang, Jiakui; Zhang, Xiang; Li, Yifan; Wang, Jingtao

    2016-08-10

    Conductive polymer electrolyte membranes are increasingly attractive for a wide range of applications in hydrogen-relevant devices, for instance hydrogen fuel cells. In this study, two-dimensional Ti3C2Tx, a typical representative of the recently developed MXene family, is synthesized and employed as a universal filler for its features of large specific surface area, high aspect ratio, and sufficient terminated -OH groups. The Ti3C2Tx is incorporated into polymer matrix to explore its function on membrane microstructure and proton conduction property. Both phase-separated (acidic Nafion and sulfonated poly(ether ether ketone)) and non-phase-separated (basic chitosan) polymers are utilized as membrane matrixes. The microstructures, physicochemical properties, and proton conduction properties of the membranes are extensively investigated. It is demonstrated that Ti3C2Tx generates significant promotion effect on proton conduction of the composite membrane by facilitating both vehicle-type and Grotthuss-type proton transfer, yielding several times increased proton conductivity for every polymer-based composite membrane under various conditions, and the composite membrane achieves elevated hydrogen fuel cell performance. The stable Ti3C2Tx also reinforces the thermal and mechanical stabilities of these composite membranes. Since the MXene family includes more than 70 members, this exploration is expected to open up new perspectives for expanding their applications, especially as membrane modifiers and proton conductors. PMID:27430190

  8. Identification of gamma-ray emission from 3C345 and NRAO512

    CERN Document Server

    Schinzel, F K; D'Ammando, F; Burnett, T H; Max-Moerbeck, W; Cheung, C C; Fegan, S J; Casandjian, J M; Reyes, L C; Villata, M; Raiteri, C M; Agudo, I; Calle, O J A Bravo; Carosati, D; Casas, R; Gomez, J L; Gurwell, M A; Hsiao, H Y; Jorstad, S G; Kimeridze, G; Konstantinova, T S; Kopatskaya, E N; Koptelova, E; Kurtanidze, O M; Kurtanidze, S O; Larionov, V M; Larionova, E G; Larionova, L V; Marscher, A P; Morozova, D A; Nikolashvili, M G; Roca-Sogorb, M; Ros, J A; Sigua, L A; Spiridonova, O; Troitsky, I S; Vlasyuk, V V; Lobanov, A P; Zensus, J A

    2011-01-01

    For more than 15 years, since the days of the Energetic Gamma-Ray Experiment Telescope (EGRET) on board the Compton Gamma-Ray Observatory (CGRO; 1991-2000), it has remained an open question why the prominent blazar 3C 345 was not reliably detected at gamma-ray energies <=20 MeV. Recently a bright gamma-ray source (0FGL J1641.4+3939/1FGL J1642.5+3947), potentially associated with 3C 345, was detected by the Large Area Telescope (LAT) on Fermi. Multiwavelength observations from radio bands to X-rays (mainly GASP-WEBT and Swift) of possible counterparts (3C 345, NRAO 512, B3 1640+396) were combined with 20 months of Fermi-LAT monitoring data (August 2008 - April 2010) to associate and identify the dominating gamma-ray emitting counterpart of 1FGL J1642.5+3947. The source 3C 345 is identified as the main contributor for this gamma-ray emitting region. However, after November 2009 (15 months), a significant excess of photons from the nearby quasar NRAO 512 started to contribute and thereafter was detected with ...

  9. Semipolar (202̅3) nitrides grown on 3C-SiC/(001) Si substrates

    Science.gov (United States)

    Dinh, Duc V.; Presa, S.; Akhter, M.; Maaskant, P. P.; Corbett, B.; Parbrook, P. J.

    2015-12-01

    Heteroepitaxial growth of GaN buffer layers on 3C-SiC/(001) Si templates (4°-offcut towards [110]) by metalorganic vapour phase epitaxy has been investigated. High-temperature grown Al0.5Ga0.5N/AlN interlayers were employed to produce a single (202̅3) GaN surface orientation. Specular crack-free GaN layers showed undulations along [11̅0]{}3{{C}-{SiC}/{Si}} with a root mean square roughness of about 13.5 nm (50 × 50 μm2). The orientation relationship determined by x-ray diffraction (XRD) was found to be [1̅21̅0]GaN ∥[11̅0]{}3{{C}-{SiC}/{Si}} and [3̅034]GaN ∥[110]3C - SiC/Si . Low-temperature photoluminescence (PL) and XRD measurements showed the presence of basal-plane stacking faults in the layers. PL measurements of (202̅3) multiple-quantum-well and light-emitting diode structures showed uniform luminescence at about 500 nm emission wavelength. A small peak shift of about 3 nm was observed in the electroluminescence when the current was increased from 5 to 50 mA (25-250 A cm-2).

  10. X-ray Emission from the Radio Jet in 3C 120

    DEFF Research Database (Denmark)

    Harris, D. E.; Hjorth, J.; Sadun, A. C.;

    1999-01-01

    We report the discovery of X-ray emission from a radio knot at a projected distance of 25" from the nucleus of the Seyfert galaxy, 3C 120. The data were obtained with the ROSAT High Resolution Imager (HRI). Optical upper limits for the knot preclude a simple power law extension of the radio spect...

  11. CW-OSL measurement protocols using optical fibre Al2O3:C dosemeters

    DEFF Research Database (Denmark)

    Edmund, J.M.; Andersen, C.E.; Marckmann, C.J.;

    2006-01-01

    A new system for in vivo dosimetry during radiotherapy has been introduced. Luminescence signals from a small crystal of carbon-doped aluminium oxide (Al2O3:C) are transmitted through an optical fibre cable to an instrument that contains optical filters, a photomultiplier tube and a green (532 nm...

  12. 40 CFR Appendix A-2 to Part 60 - Test Methods 2G through 3C

    Science.gov (United States)

    2010-07-01

    ... (if used) shall be coupled with a data display device (such as a digital panel meter, personal... personal computer with data capture software) that has the ability to compute and retain the appropriate... 40 Protection of Environment 7 2010-07-01 2010-07-01 true Test Methods 2G through 3C A Appendix...

  13. Temperature dependence of the Al2O3:C response in medical luminescence dosimetry

    DEFF Research Database (Denmark)

    Edmund, Jens Morgenthaler; Andersen, Claus Erik

    2007-01-01

    Over the last years, attention has been given to applications of Al2O3:C in space and medical dosimetry. One such application is in vivo dose verification in radiotherapy of cancer patients and here we investigate the temperature effects on the radioluminescence (RL) and optically stimulated...

  14. Evidence-Based Nursing of the 3C Therapeutic Regimen for Type 1 Diabetes.

    Science.gov (United States)

    Wu, Jianya; Zou, Ling

    2015-05-01

    The aim of this study is to explore the efficacy of the 3C therapeutic regimen for type 1 diabetes. Thirty-nine patients with type 1 diabetes, who were hospitalized from January 2013 to April 2014, were included to receive 3C therapeutic regimen. Evidence-based nursing was performed in the treatment period and the efficacy was observed 6 days after therapy. Six days after the administration of the 3C therapeutic regimen, the fasting glucose levels in all 39 patients were controlled to be 4.4-6.0 mmol/L and 2h-postprandial glucose levels to be 4.4-7.8 mmol/L. Three patients had a glucose level <3.9 mmol/L, which was corrected after adjusting the dose of insulin infusion. Evidence-based nursing was provided in the treatment period and no nursing-associated complication occurred. All patients were satisfied with the nursing service. The efficacy of the 3C therapeutic regimen for type 1 diabetes is satisfactory. The evidence-based nursing can help to ensure the efficacy and improve the quality of nursing service. PMID:25424358

  15. Allylic and benzylic sp3 C-H oxidation in water.

    Science.gov (United States)

    Ang, Wei Jie; Lam, Yulin

    2015-01-28

    A copper-catalyzed method for the oxidation of allylic and benzylic sp(3) C-H by aqueous tert-butyl hydroperoxide (T-Hydro) in water using a recyclable fluorous ligand has been developed. The reaction procedure is tolerant to additional functional groups and the fluorous ligand could be reused with little loss of catalytic activity. PMID:25412371

  16. WSRT AND VLA OBSERVATIONS OF HI IN THE DIRECTION OF 3C-10

    NARCIS (Netherlands)

    SCHWARZ, UJ; GOSS, WM; KALBERLA, PM; BENAGLIA, P

    1995-01-01

    Westerbork Synthesis Radio Telescope HI observations combined with Effelsberg 100 m antenna observations have been made of the galactic supernova remnant 3C 10 (Tycho) with an angular resolution similar or equal to 50 '' and a velocity resolution of 0.62 km s(-1) and with the VLA with resolutions of

  17. Fast outflow of Hi in starburst radio galaxy 3C 293

    NARCIS (Netherlands)

    Emonts, B; van der Hulst, T; Morganti, R; Oosterloo, T; Tadhunter, C; Holt, J; Wills, K; Aalto, S; Huttemeister, S; Pedlar, A

    2004-01-01

    We detect a fast outflow of gas in the central region of the nearby starburst radio galaxy 3C 293. The outflow is detected both in the optical emission lines of ionized gas as well as in HI absorption against the radio continuum. The broad HI absorption feature (observed with the recently upgraded W

  18. The corona of the broad-line radio galaxy 3C 390.3

    DEFF Research Database (Denmark)

    Lohfink, A. M.; Ogle, P.; Tombesi, F.;

    2015-01-01

    -UVOT data, we find indications that the compactness of the corona allows only a small fraction of the total UV/optical flux to be Comptonized. Our analysis of the reprocessed emission show that 3C 390.3 only has a small amount of reflection (R ~ 0.3), and of that the vast majority is from distant neutral...

  19. 3C 273 with NuSTAR: Unveiling the Active Galactic Nucleus

    DEFF Research Database (Denmark)

    Madsen, Kristin K.; Fürst, Felix; Walton, Dominic J.;

    2015-01-01

    We present results from a 244 ks NuSTAR observation of 3C 273 obtained during a cross-calibration campaign with the Chandra, INTEGRAL, Suzaku, Swift, and XMM-Newton observatories. We show that the spectrum, when fit with a power-law model using data from all observatories except INTEGRAL over the...

  20. LM-3C:Breakthrough in Launch Technology for Deep Space Exploration

    Institute of Scientific and Technical Information of China (English)

    Li Dan; Hu Wei; Wei Yuanming

    2010-01-01

    @@ One of the main objectives ofthe Chang'e 2 mission was to demonstrate the direct Earthmoon transfer orbit launch technology.On October 1, 2010, a LM-3C launch vehicle sent the Chang'e 2 into its preset orbit accurately, demonstrating that China has made a breakthrough in launch technology for deep space exploration.

  1. Optical Monitoring of 3C 66A in 1994–2008

    Indian Academy of Sciences (India)

    J. Tao; J. H. Fan; B. C. Qian; H. J. Pan

    2011-03-01

    3C 66A is one of the most interesting blazar. Our monitoring was carried out with a 1.56-m telescope of the Shanghai Astronomical Observatory (SHAO) from 13 December 1994 to 9 November 2008. Some peaks and gradual brightening of the source up to three times were observed.

  2. Frequency dependent core shifts and parameter estimation for the blazar 3C 454.3

    CERN Document Server

    Mohan, P; Mangalam, A; Gupta, Alok C; Wiita, Paul J; Volvach, A E; Aller, M F; Aller, H D; Gu, M F; Lahteenmaki, A; Tornikoski, M; Volvach, L N

    2015-01-01

    We study the core shift effect in the parsec scale jet of the blazar 3C 454.3 using the 4.8 GHz - 36.8 GHz radio light curves obtained from three decades of continuous monitoring. From a piecewise Gaussian fit to each flare, time lags $\\Delta t$ between the observation frequencies $\

  3. A Deep XMM-Newton Observation of the Quasar 3C 287

    CERN Document Server

    Salvesen, G; Cackett, E; Siemiginowska, A

    2008-01-01

    We report on an XMM-Newton observation of the z=1.055 quasar and Giga-hertz Peaked Spectrum (GPS) source 3C 287. Our 62.3 ksec observation provides an exceptional X-ray view of a prominent member of this important subclass of active galactic nuclei (AGN). The X-ray spectra of 3C 287 are consistent with a simple absorbed power-law with a spectral index of Gamma = 1.72 +/- 0.02. Our fits imply a bolometric luminosity of L = 5.8 +/- 0.2 E+45 erg/s over the 0.3-10.0 keV band; this gives a mass lower limit of M > 4.6 E+7 Msun, assuming X-rays contribute 10% of the bolometric luminosity and radiation at the Eddington limit. Iron emission lines are common in the X-ray spectra of many AGN, but the observed spectra appear to rule out strong emission lines in 3C 287. The simple power-law spectrum and absence of strong emission lines may support a picture where our line of sight intersects a relativistic jet. Milliarcsecond radio imaging of 3C 287 appears to support this interpretation. We discuss our results in the con...

  4. A thin layer fiber-coupled luminescence dosimeter based on Al2O3:C

    DEFF Research Database (Denmark)

    Klein, F.A.; Greilich, Steffen; Andersen, Claus Erik;

    2011-01-01

    In this paper we present a fiber-coupled luminescent Al2O3:C dosimeter probe with high spatial resolution (0.1 mm). It is based on thin layers of Al2O3:C crystal powder and a UV-cured acrylate monomer composition. The fabrication of the thin layers is described in detail. No influence of the intr......In this paper we present a fiber-coupled luminescent Al2O3:C dosimeter probe with high spatial resolution (0.1 mm). It is based on thin layers of Al2O3:C crystal powder and a UV-cured acrylate monomer composition. The fabrication of the thin layers is described in detail. No influence...... without sensitivity corrections. For protons, a relative luminescence efficiency hHCP of 0.715 0.014 was found in the Bragg peak. For carbon ions, a value of 0.498 0.001 was found in the entrance channel, 0.205 0.015 in the Bragg peak, and a mean of 0.413 0.050 in the tail region. The mean range...

  5. The temperature dependence of optically stimulated luminescence from α-Al2O3:C

    DEFF Research Database (Denmark)

    Markey, B.G.; McKeever, S.W.S.; Akselrod, M.S.;

    1996-01-01

    The results of experimental measurements and computer simulations on optically stimulated luminescence (OSL) from alpha-Al2O3:C are described. The intensity of the OSL observed during illumination of irradiated specimens with visible light is temperature dependent. Optical stimulation is observed...

  6. Allostery in trypsin-like proteases suggests new therapeutic strategies.

    Science.gov (United States)

    Gohara, David W; Di Cera, Enrico

    2011-11-01

    Trypsin-like proteases (TLPs) are a large family of enzymes responsible for digestion, blood coagulation, fibrinolysis, development, fertilization, apoptosis and immunity. A current paradigm posits that the irreversible transition from an inactive zymogen to the active protease form enables productive interaction with substrate and catalysis. Analysis of the entire structural database reveals two distinct conformations of the active site: one fully accessible to substrate (E) and the other occluded by the collapse of a specific segment (E*). The allosteric E*-E equilibrium provides a reversible mechanism for activity and regulation in addition to the irreversible zymogen to protease conversion and points to new therapeutic strategies aimed at inhibiting or activating the enzyme. In this review, we discuss relevant examples, with emphasis on the rational engineering of anticoagulant thrombin mutants.

  7. Tobacco Etch Virus protease: A shortcut across biotechnologies.

    Science.gov (United States)

    Cesaratto, Francesca; Burrone, Oscar R; Petris, Gianluca

    2016-08-10

    About thirty years ago, studies on the RNA genome of Tobacco Etch Virus revealed the presence of an efficient and specific protease, called Tobacco Etch Virus protease (TEVp), that was part of the Nuclear Inclusion a (NIa) enzyme. TEVp is an efficient and specific protease of 27kDa that has become a valuable biotechnological tool. Nowadays TEVp is a unique endopeptidase largely exploited in biotechnology from industrial applications to in vitro and in vivo cellular studies. A number of TEVp mutants with different rate of cleavage, stability and specificity have been reported. Similarly, a panel of different target cleavage sites, derived from the canonical ENLYFQ-G/S site, has been established. In this review we describe these aspects of TEVp and some of its multiple applications. A particular focus is on the use and molecular biology of TEVp in living cells and organisms.

  8. Peptide synthesis in neat organic solvents with novel thermostable proteases.

    Science.gov (United States)

    Toplak, Ana; Nuijens, Timo; Quaedflieg, Peter J L M; Wu, Bian; Janssen, Dick B

    2015-06-01

    Biocatalytic peptide synthesis will benefit from enzymes that are active at low water levels in organic solvent compositions that allow good substrate and product solubility. To explore the use of proteases from thermophiles for peptide synthesis under such conditions, putative protease genes of the subtilase class were cloned from Thermus aquaticus and Deinococcus geothermalis and expressed in Escherichia coli. The purified enzymes were highly thermostable and catalyzed efficient peptide bond synthesis at 80°C and 60°C in neat acetonitrile with excellent conversion (>90%). The enzymes tolerated high levels of N,N-dimethylformamide (DMF) as a cosolvent (40-50% v/v), which improved substrate solubility and gave good conversion in 5+3 peptide condensation reactions. The results suggest that proteases from thermophiles can be used for peptide synthesis under harsh reaction conditions.

  9. Substrate specificity of the ubiquitin and Ubl proteases

    Science.gov (United States)

    Ronau, Judith A; Beckmann, John F; Hochstrasser, Mark

    2016-01-01

    Conjugation and deconjugation of ubiquitin and ubiquitin-like proteins (Ubls) to cellular proteins are highly regulated processes integral to cellular homeostasis. Most often, the C-termini of these small polypeptides are attached to lysine side chains of target proteins by an amide (isopeptide) linkage. Deubiquitinating enzymes (DUBs) and Ubl-specific proteases (ULPs) comprise a diverse group of proteases that recognize and remove ubiquitin and Ubls from their substrates. How DUBs and ULPs distinguish among different modifiers, or different polymeric forms of these modifiers, remains poorly understood. The specificity of ubiquitin/Ubl-deconjugating enzymes for particular substrates depends on multiple factors, ranging from the topography of specific substrate features, as in different polyubiquitin chain types, to structural elements unique to each enzyme. Here we summarize recent structural and biochemical studies that provide insights into mechanisms of substrate specificity among various DUBs and ULPs. We also discuss the unexpected specificities of non-eukaryotic proteases in these families. PMID:27012468

  10. Protease-triggered siRNA delivery vehicles.

    Science.gov (United States)

    Rozema, David B; Blokhin, Andrei V; Wakefield, Darren H; Benson, Jonathan D; Carlson, Jeffrey C; Klein, Jason J; Almeida, Lauren J; Nicholas, Anthony L; Hamilton, Holly L; Chu, Qili; Hegge, Julia O; Wong, So C; Trubetskoy, Vladimir S; Hagen, Collin M; Kitas, Eric; Wolff, Jon A; Lewis, David L

    2015-07-10

    The safe and efficacious delivery of membrane impermeable therapeutics requires cytoplasmic access without the toxicity of nonspecific cytoplasmic membrane lysis. We have developed a mechanism for control of cytoplasmic release which utilizes endogenous proteases as a trigger and results in functional delivery of small interfering RNA (siRNA). The delivery approach is based on reversible inhibition of membrane disruptive polymers with protease-sensitive substrates. Proteolytic hydrolysis upon endocytosis restores the membrane destabilizing activity of the polymers thereby allowing cytoplasmic access of the co-delivered siRNA. Protease-sensitive polymer masking reagents derived from polyethylene glycol (PEG), which inhibit membrane interactions, and N-acetylgalactosamine, which targets asialoglycoprotein receptors on hepatocytes, were synthesized and used to formulate masked polymer-siRNA delivery vehicles. The size, charge and stability of the vehicles enable functional delivery of siRNA after subcutaneous administration and, with modification of the targeting ligand, have the potential for extrahepatic targeting.

  11. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    Science.gov (United States)

    Witzmann, F. A.; Troup, J. P.; Fitts, R. H.

    1982-01-01

    The effect of hind-limb immobilization on selected Iysosomal enzyme activities was studied in rat hing-limb muscles composed primarily of type 1. 2A, or 2B fibers. Following immobilization, acid protease and acid phosphatase both exhibited signifcant increases in their activity per unit weight in all three fiber types. Acid phosphatase activity increased at day 14 of immobilization in the three muscles and returned to control levels by day 21. Acid protease activity also changed biphasically, displaying a higher and earlier rise than acid phosphatase. The pattern of change in acid protease, but not acid phosphatase, closely parallels observed muscle wasting. The present data therefore demonstrate enhanced proteolytic capacity of all three fiber types early during muscular atrophy. In addition, the data suggest a dependence of basal hydrolytic and proteolytic activities and their adaptive response to immobilization on muscle fiber composition.

  12. Cathepsin G, a Neutrophil Protease, Induces Compact Cell-Cell Adhesion in MCF-7 Human Breast Cancer Cells

    Directory of Open Access Journals (Sweden)

    Tomoya Kudo

    2009-01-01

    Full Text Available Cathepsin G is a serine protease secreted by activated neutrophils that play a role in the inflammatory response. Because neutrophils are known to be invading leukocytes in various tumors, their products may influence the characteristics of tumor cells such as the growth state, motility, and the adhesiveness between cells or the extracellular matrix. Here, we demonstrate that cathepsin G induces cell-cell adhesion of MCF-7 human breast cancer cells resulting from the contact inhibition of cell movement on fibronectin but not on type IV collagen. Cathepsin G subsequently induced cell condensation, a very compact cell colony, resulting due to the increased strength of E-cadherin-mediated cell-cell adhesion. Cathepsin G action is protease activity-dependent and was inhibited by the presence of serine protease inhibitors. Cathepsin G promotes E-cadherin/catenin complex formation and Rap1 activation in MCF-7 cells, which reportedly regulates E-cadherin-based cell-cell junctions. Cathepsin G also promotes E-cadherin/protein kinase D1 (PKD1 complex formation, and Go6976, the selective PKD1 inhibitor, suppressed the cathepsin G-induced cell condensation. Our findings provide the first evidence that cathepsin G regulates E-cadherin function, suggesting that cathepsin G has a novel modulatory role against tumor cell-cell adhesion.

  13. Structural Basis of Why Nelfinavir-Resistant D30N Mutant of HIV-1 Protease Remains Susceptible to Saquinavir.

    Science.gov (United States)

    Prashar, Vishal; Bihani, Subhash C; Ferrer, Jean-Luc; Hosur, Madhusoodan V

    2015-09-01

    Although anti-HIV-1 protease drugs nelfinavir (NFV) and saquinavir (SQV) share common functional groups, D30N is a major resistance mutation against NFV but remains susceptible to SQV. We have determined the crystal structure of D30N mutant-tethered HIV-1 protease in complex with SQV to 1.79 Å resolution. Structural analysis showed that SQV forms two direct hydrogen bonds with the main chain atoms of the residues Asp29 and Asp30 that are not observed in the D30N-NFV complex. Apart from maintaining these two main chain hydrogen bonds, the P2-asparagine of SQV forms an additional hydrogen bond to the mutated side chain of the residue 30. These could be the reasons why D30N is not a drug resistance mutation against SQV. This structure supports the previous studies showing that the interactions between a potential inhibitor and backbone atoms of the enzyme are important to maintain potency against drug-resistant HIV-1 protease. PMID:25487655

  14. Trichuris suis: thiol protease activity from adult worms.

    Science.gov (United States)

    Hill, D E; Sakanari, J A

    1997-01-01

    Trichuris suis, the whipworm of swine, causes anemia, weight loss, anorexia, mucohemorrhagic diarrhea, and death in heavy infections. A zinc metalloprotease has been suggested to play a role in the severe enteric pathology associated with infection and the infiltration of opportunistic bacteria into deeper tissues in the swine colon. In this study, a thiol protease from gut extracts of adult T. suis and from excretory/secretory components (E/S) of adult worms was characterized using fluorogenic peptide substrates and protein substrate gels. The protease cleaved the fluorogenic substrate Z-Phe-Arg-AMC, and this cleavage was completely inhibited by the thiol protease inhibitors E-64, leupeptin, Z-Phe-Ala-CH2F, and Z-Phe-Arg-CH2F. Gelatin substrate gels and fluorescence assays using both the gut and the stichosome extracts and E/S revealed enhanced activity when 2 mM dithiothreitol or 5 mM cysteine was included in the incubation buffer, and optimal activity was seen over a pH range of 5.5 to 8.5. Incubation of gut extracts or E/S material with inhibitors of aspartic, serine, or metalloproteases had no effect on the cleavage of Z-Phe-Arg-AMC. Thiol protease activity was found in extracts of gut tissue but not in the extracts of stichocytes of adult worms. N-terminal amino acid sequencing of the protease revealed sequence homologies with cathepsin B-like thiol protease identified from parasitic and free-living nematodes. PMID:9024202

  15. Galectin-3 cDNA Diversity in Oryctolagus cuniculus%兔半乳糖凝聚素-3 cDNA分子多样性分析

    Institute of Scientific and Technical Information of China (English)

    方恩浩; 张珊珊; 刘倩芸; 周建钟; 杨仙玉

    2015-01-01

    半乳糖凝聚素-3 (galectin-3, Gal-3)是一种多功能蛋白,参与细胞增殖、分化、凋亡、血管发生及肿瘤发生与转移等多种生命活动过程.前期工作表明一些两栖类动物存在个体水平的gal-3 cDNA分子多样性.鉴于哺乳动物尚缺乏这方面的研究,选择新西兰兔(Oryctolagus cuniculus)为实验材料,制备3只不同个体的肝脏第一链cDNA,通过DNA聚合酶链式反应扩增gal-3开放阅读框(open reading frame,ORF)并进行克隆测序.结果表明,兔gal-3 ORF中存在5个单核苷酸多态性(single nucleotide polymorphism,SNP)位点,导致11个不同的ORF,其中1个与GenBank注册的序列完全相同,另外10个均为本研究首次发现.从1~3号个体的肝脏分别获得6、6、2条不同的兔gal-3 ORF,揭示兔肝脏存在个体水平的gal-3 cDNA分子多样性.

  16. [Isolation of Actinomycetes synthesizing proteases with thrombolytic activity].

    Science.gov (United States)

    Lysenko, S V; Salivonik, S M

    1988-01-01

    Proteases with the thrombolytic activity were studied in 212 strains of actinomycetes isolated from different soils of the Soviet Union. The cultures belonged to the genera Micromonospora, Nocardia and Streptomyces. Proteases were synthesized by 41% of the studied actinomycetes and some of their strains completely dissolved in vitro artificially obtained blood thrombi within 120-240 min. In the Streptomyces genus, more active strains were found in the groups Flavus, Fradia and Globisporus. The groups Olivaceus, Violaceus and Viridis had less active strains. PMID:3062331

  17. 现代社会肠道复合微生态改变与疾病--消化蛋白酶灭活障碍或是关键%Changes in ComplexMicroecosystem of Gut and Pathogenesis of Diseases in Modern Society --Impaired Inactivation of Digestive Proteases May Be the Key Event

    Institute of Scientific and Technical Information of China (English)

    秦孝发

    2015-01-01

    Hygiene Hypothesis”has been gradually formed, attributing this change to the reduced exposure to microbes such as bacteria and the resultant aberrant immune system. However, the exact mechanism remains elusive. As most of the bacteria exist in intestine, the relationship between changes in the complexmicroecosystem of gut and these diseases has drawn more and more attention. Many studies revealed that many autoimmune, allergic and metabolic diseases, and diseases of the gut, lung, cardiovascular, liver, kidney, skin, and joint, diabetes, obesity, morbidity of the nervous system, and even some cancer are associated with increased gut permeability. As the gut contains bacteria and endotoxin that can kill the host hundreds or thousand times over,leakage of this highly dangerous internal contaminating source could be deifnitely more devastating than environmental pollution, leading to damage of every organ inside the body, and causing or exacerbating many diseases. Therefore it would be critical to elucidate the mechanism behind the increased intestinal permeability. I have found that digestive proteases can be inactivated be unconjugated bilirubin. Deconjugation of bilirubin depends on the enzyme mainly existed in gut bacteria. Based on the large amount of evidence collected, I suspected that reduction in gut bacteria along with improved hygiene and inhibition by some dietary chemicals in modern society and the resultant impaired inactivation of digestive proteases, damage ingut tissue and barrier, increased inifltration of bacteria and other luminal toxicants may have played critical role in the pathogenesis of diseases such as IBD. Thus, impaired inactivation of digestive proteases due to reduction of gut bacteria may be the key event of the problem. This paper aims to make a discussion on this.

  18. PENGEMPUKAN DAGING DENGAN ENZIM PROTEASE TANAMAN BIDURI (Calotropis gigantea [Meat Tenderization using Protease of Biduri Plant (Calotropis gigantea

    Directory of Open Access Journals (Sweden)

    Erni Sofia Murtini1

    2003-12-01

    Full Text Available Tenderness is the main attribute quality of meat, which influences consumer acceptability. Protease enzyme (like papain, bromelin and ficin are known to be used for improving tenderness of meat trough degradation of the protein. Biduri plant (Calotropis gigantea contains protease enzyme in its latex or the young tissue (0-20 cm plant tip. After isolation of crude enzyme using ammonium sulphate, the enzyme was the applied to tenderise meat at concentrations 0 ; 0,25; 0,5; 0,75 and 1,0%. The result showed that concentration of protease enzyme affected to meat tenderness that determined by compression test and tensile strength. The enzyme (0.5% was enough to tenderise meat indicated by decreasing its compression test value to 201,160 N 9 from control of 228,582 N and tensile strength value to 4,618 N (from control 9,588N

  19. ISOLASI DAN KARAKTERISASI PROTEASE DARI BAKTERI TANAH RAWA INDRALAYA, SUMATERA SELATAN [Isolation and Characterization of Proteases from Indralaya Soil Swamp Bacteria,South Sumatera

    Directory of Open Access Journals (Sweden)

    Ace Baehaki*

    2011-06-01

    Full Text Available In an effort of obtaining indigenous protease producing bacteria, screening for bacterial protease was conducted from samples collected from Indralaya soil swamp, South Sumatera. Three of 31 colonies showed high protease activity with proteolytic index >1.00. T1S1 produced enzyme with the highest activity. The crude enzyme activity after 48 hours of incubation was 0.391 IU/ml. The optimum pH of the extracelull proteases from T1S1, T3S2 and T3S3 were 8.0, 8.0, and 7.5, respectively. The optimum temperature of T1S1, T3S2 and T3S3 proteases were 40, 50, and 500C, respectively. All metal ions tested (Na+, K+, Mn2+, Zn2+ and Fe2+ inhibited proteases except Fe2+ which activatesthe T3S3 protease at 5 mM. EDTA (1 and 5 mM inhibited all proteases. Study on the effect of metals ion and spesific inhibitors indicated that all protease are metaloprotease. Molecular weights was determined using SDS-PAGE and zymogram technique. The molecular weight of T1S1 proteases was 121 kD,T3S2 proteaseswere 51, 71, and 119 kD whereas T3S3 proteaseswere 49, 70, and 116 kD.

  20. Semaphorin 3C Released from a Biocompatible Hydrogel Guides and Promotes Axonal Growth of Rodent and Human Dopaminergic Neurons.

    Science.gov (United States)

    Carballo-Molina, Oscar A; Sánchez-Navarro, Andrea; López-Ornelas, Adolfo; Lara-Rodarte, Rolando; Salazar, Patricia; Campos-Romo, Aurelio; Ramos-Mejía, Verónica; Velasco, Iván

    2016-06-01

    Cell therapy in experimental models of Parkinson's disease replaces the lost dopamine neurons (DAN), but we still need improved methods to guide dopaminergic axons (DAx) of grafted neurons to make proper connections. The protein Semaphorin 3C (Sema3C) attracts DAN axons and enhances their growth. In this work, we show that the hydrogel PuraMatrix, a self-assembling peptide-based matrix, incorporates Sema3C and releases it steadily during 4 weeks. We also tested if hydrogel-delivered Sema3C attracts DAx using a system of rat midbrain explants embedded in collagen gels. We show that Sema3C released by this hydrogel attracts DAx, in a similar way to pretectum, which is known to attract growing DAN axons. We assessed the effect of Sema3C on the growth of DAx using microfluidic devices. DAN from rat midbrain or those differentiated from human embryonic stem cells showed enhanced axonal extension when exposed to hydrogel-released Sema3C, similar to soluble Sema3C. Notably, DAN of human origin express the cognate Sema3C receptors, Neuropilin1 and Neuropilin2. These results show that PuraMatrix is able to incorporate and release Sema3C, and such delivery guides and promotes the axonal growth of DAN. This biocompatible hydrogel might be useful as a Sema3C carrier for in vivo studies in parkinsonian animal models. PMID:27174503