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Sample records for 3c protease 3cpro

  1. The Coxsackievirus B 3Cpro Protease Cleaves MAVS and TRIF to Attenuate Host Type I Interferon and Apoptotic Signaling

    PubMed Central

    Mukherjee, Amitava; Morosky, Stefanie A.; Delorme-Axford, Elizabeth; Dybdahl-Sissoko, Naomi; Oberste, M. Steven; Wang, Tianyi; Coyne, Carolyn B.

    2011-01-01

    The host innate immune response to viral infections often involves the activation of parallel pattern recognition receptor (PRR) pathways that converge on the induction of type I interferons (IFNs). Several viruses have evolved sophisticated mechanisms to attenuate antiviral host signaling by directly interfering with the activation and/or downstream signaling events associated with PRR signal propagation. Here we show that the 3Cpro cysteine protease of coxsackievirus B3 (CVB3) cleaves the innate immune adaptor molecules mitochondrial antiviral signaling protein (MAVS) and Toll/IL-1 receptor domain-containing adaptor inducing interferon-beta (TRIF) as a mechanism to escape host immunity. We found that MAVS and TRIF were cleaved in CVB3-infected cells in culture. CVB3-induced cleavage of MAVS and TRIF required the cysteine protease activity of 3Cpro, occurred at specific sites and within specialized domains of each molecule, and inhibited both the type I IFN and apoptotic signaling downstream of these adaptors. 3Cpro-mediated MAVS cleavage occurred within its proline-rich region, led to its relocalization from the mitochondrial membrane, and ablated its downstream signaling. We further show that 3Cpro cleaves both the N- and C-terminal domains of TRIF and localizes with TRIF to signalosome complexes within the cytoplasm. Taken together, these data show that CVB3 has evolved a mechanism to suppress host antiviral signal propagation by directly cleaving two key adaptor molecules associated with innate immune recognition. PMID:21436888

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

    PubMed Central

    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

  3. Synthesis and evaluation of peptidyl Michael acceptors that inactivate human rhinovirus 3C protease and inhibit virus replication.

    PubMed

    Kong, J S; Venkatraman, S; Furness, K; Nimkar, S; Shepherd, T A; Wang, Q M; Aubé, J; Hanzlik, R P

    1998-07-01

    Human rhinovirus, the chief cause of the common cold, contains a positive-sense strand of RNA which is translated into a large polyprotein in infected cells. Cleavage of the latter to produce the mature viral proteins required for replication is catalyzed in large part by a virally encoded cysteine proteinase (3Cpro) which is highly selective for -Q approximately GP- cleavage sites. We synthesized peptidyl derivatives of vinylogous glutamine or methionine sulfone esters (e.g., Boc-Val-Leu-Phe-vGln-OR: R = Me, 1; R = Et, 2) and evaluated them as inhibitors of HRV-14 3C protease (3Cpro). Compounds 1 and 2 and several related tetra- and pentapeptide analogues rapidly inactivated 3Cpro with submicromolar IC50 values. Electrospray mass spectrometry confirmed the expected 1:1 stoichiometry of 3Cpro inactivation by 1, 2, and several other analogues. Compound 2 also proved to be useful for active site titration of 3Cpro, which has not been possible heretofore because of the lack of a suitable reagent. In contrast to 1, 2, and congeners, peptidyl Michael acceptors lacking a P4 residue have greatly reduced or negligible activity against 3Cpro, consistent with previously established structure-activity relationships for 3Cpro substrates. Hydrolysis of the P1 vinylogous glutamine ester to a carboxylic acid also decreased inhibitory activity considerably, consistent with the decreased reactivity of acrylic acids vs acrylic esters as Michael acceptors. Incorporating a vinylogous methionine sulfone ester in place of the corresponding glutamine derivative in 1 also reduced activity substantially. Compounds 1 and 2 and several of their analogues inhibited HRV replication in cell culture by 50% at low micromolar concentrations while showing little or no evidence of cytotoxicity at 10-fold higher concentrations. Peptidyl Michael acceptors and their analogues may prove useful as therapeutic agents for pathologies involving cysteine proteinase enzymes.

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

    PubMed

    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

  5. Peptidyl Aldehyde NK-1.8k Suppresses Enterovirus 71 and Enterovirus 68 Infection by Targeting Protease 3C

    PubMed Central

    Wang, Yaxin; Yang, Ben; Zhai, Yangyang; Rao, Zihe

    2015-01-01

    Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 μM) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3Cpro), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3Cpro. The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy. PMID:25691647

  6. Identification of hepatitis A virus non-structural protein 2B and its release by the major virus protease 3C.

    PubMed

    Gosert, R; Cassinotti, P; Siegl, G; Weitz, M

    1996-02-01

    The RNA genome of hepatitis A virus (HAV) encodes a giant polyprotein that is putatively cleaved proteolytically into four structural and seven non-structural proteins. So far, most of the proposed non-structural proteins and their respective cleavage sites have not been identified. A vaccinia virus recombinant (vRGORF) containing the complete HAV ORF under the control of the bacteriophage T7 promoter was used to express HAV in recombinant animal cells (BT7-H) that constitutively expressed T7 DNA-dependent RNA polymerase. A HAV-specific 27.5 kDa expression product was identified as peptide 2B. The 27.5 kDa 2B antigen was also found in HAV-infected MRC-5 cells. The N-terminal amino acid residues of the new peptide 2B are Ala-Lys-Ile-Ser-Leu-Phe and polyprotein cleavage between 2A and 2B occurred at amino acids 836-837 (Gln-Ala). Furthermore, heterologous expression in the same system of regions P1-P2 and of the protease 3C (3Cpro) gene, showed that P1-P2 polyprotein is not cleaved autocatalytically but by 3Cpro. Hence, 3Cpro is effective in cleaving the polyprotein 2A-2B junction.

  7. Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy.

    PubMed

    Miles, Linde A; Brennen, W Nathaniel; Rudin, Charles M; Poirier, John T

    2015-01-01

    The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M(-1)s(-1)), was further optimized by a P2' N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M(-1)s(-1)). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development. PMID:26069962

  8. Seneca Valley Virus 3Cpro Substrate Optimization Yields Efficient Substrates for Use in Peptide-Prodrug Therapy.

    PubMed

    Miles, Linde A; Brennen, W Nathaniel; Rudin, Charles M; Poirier, John T

    2015-01-01

    The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 ± 183 M(-1)s(-1)), was further optimized by a P2' N→P substitution yielding L/VP4.1 (kcat/KM = 17446 ± 2203 M(-1)s(-1)). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development.

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

    PubMed

    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.

  10. Development of potent inhibitors of the coxsackievirus 3C protease

    SciTech Connect

    Lee, Eui Seung; Lee, Won Gil; Yun, Soo-Hyeon; Rho, Seong Hwan; Im, Isak; Yang, Sung Tae; Sellamuthu, Saravanan; Lee, Yong Jae; Kwon, Sun Jae; Park, Ohkmae K.; Jeon, Eun-Seok; Park, Woo Jin . E-mail: wjpark@gist.ac.kr; Kim, Yong-Chul . E-mail: yongchul@gist.ac.kr

    2007-06-22

    Coxsackievirus B3 (CVB3) 3C protease (3CP) plays essential roles in the viral replication cycle, and therefore, provides an attractive therapeutic target for treatment of human diseases caused by CVB3 infection. CVB3 3CP and human rhinovirus (HRV) 3CP have a high degree of amino acid sequence similarity. Comparative modeling of these two 3CPs revealed one prominent distinction; an Asn residue delineating the S2' pocket in HRV 3CP is replaced by a Tyr residue in CVB3 3CP. AG7088, a potent inhibitor of HRV 3CP, was modified by substitution of the ethyl group at the P2' position with various hydrophobic aromatic rings that are predicted to interact preferentially with the Tyr residue in the S2' pocket of CVB3 3CP. The resulting derivatives showed dramatically increased inhibitory activities against CVB3 3CP. In addition, one of the derivatives effectively inhibited the CVB3 proliferation in vitro.

  11. Potent inhibition of feline coronaviruses with peptidyl compounds targeting coronavirus 3C-like protease.

    PubMed

    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.

  12. Roles of the Picornaviral 3C Proteinase in the Viral Life Cycle and Host Cells

    PubMed Central

    Sun, Di; Chen, Shun; Cheng, Anchun; Wang, Mingshu

    2016-01-01

    The Picornaviridae family comprises a large group of non-enveloped viruses that have a major impact on human and veterinary health. The viral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteinases. The crucial 3C proteinases (3Cpros) of picornaviruses share similar spatial structures and it is becoming apparent that 3Cpro plays a significant role in the viral life cycle and virus host interaction. Importantly, the proteinase and RNA-binding activity of 3Cpro are involved in viral polyprotein processing and the initiation of viral RNA synthesis. In addition, 3Cpro can induce the cleavage of certain cellular factors required for transcription, translation and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Due to interactions between 3Cpro and these essential factors, 3Cpro is also involved in viral pathogenesis to support efficient infection. Furthermore, based on the structural conservation, the development of irreversible inhibitors and discovery of non-covalent inhibitors for 3Cpro are ongoing and a better understanding of the roles played by 3Cpro may provide insights into the development of potential antiviral treatments. In this review, the current knowledge regarding the structural features, multiple functions in the viral life cycle, pathogen host interaction, and development of antiviral compounds for 3Cpro is summarized. PMID:26999188

  13. A continuous assay for foot-and-mouth disease virus 3C protease activity.

    PubMed

    Jaulent, Agnès M; Fahy, Aodhnait S; Knox, Stephen R; Birtley, James R; Roqué-Rosell, Núria; Curry, Stephen; Leatherbarrow, Robin J

    2007-09-15

    Foot-and-mouth disease virus is a highly contagious pathogen that spreads rapidly among livestock and is capable of causing widespread agricultural and economic devastation. The virus genome is translated to produce a single polypeptide chain that subsequently is cleaved by viral proteases into mature protein products, with one protease, 3C(pro), carrying out the majority of the cleavages. The highly conserved nature of this protease across different viral strains and its crucial role in viral maturation and replication make it a very desirable target for inhibitor design. However, the lack of a convenient and high-throughput assay has been a hindrance in the characterization of potential inhibitors. In this article, we report the development of a continuous assay with potential for high throughput using fluorescence resonance energy transfer-based peptide substrates. Several peptide substrates containing the 3C-specific cleavage site were synthesized, varying both the positions and separation of the fluorescent donor and quencher groups. The best substrate, with a specificity constant k(cat)/K(M) of 57.6+/-2.0M(-1) s(-1), was used in inhibition assays to further characterize the protease's activity against a range of commercially available inhibitors. The inhibition profile of the enzyme showed characteristics of both cysteine and serine proteases, with the chymotrypsin inhibitor TPCK giving stoichiometric inhibition of the enzyme and allowing active site titration of the 3C(pro).

  14. Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro.

    PubMed

    Cao, Zeyu; Ding, Yue; Ke, Zhipeng; Cao, Liang; Li, Na; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

    2016-01-01

    Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro.

  15. Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro

    PubMed Central

    Cao, Zeyu; Ding, Yue; Ke, Zhipeng; Cao, Liang; Li, Na; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

    2016-01-01

    Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro. PMID:26870944

  16. Luteoloside Acts as 3C Protease Inhibitor of Enterovirus 71 In Vitro.

    PubMed

    Cao, Zeyu; Ding, Yue; Ke, Zhipeng; Cao, Liang; Li, Na; Ding, Gang; Wang, Zhenzhong; Xiao, Wei

    2016-01-01

    Luteoloside is a member of the flavonoids family that exhibits several bioactivities including anti-microbial and anti-cancer activities. However, the antiviral activity of luteoloside against enterovirus 71 (EV71) and the potential mechanism(s) responsible for this effect remain unknown. In this study, the antiviral potency of luteoloside against EV71 and its inhibitory effects on 3C protease activity were evaluated. First, we investigated the cytotoxicity of luteoloside against rhabdomyosarcoma (RD) cells, which was the cell line selected for an in vitro infection model. In a subsequent antiviral assay, the cytopathic effect of EV71 was significantly and dose-dependently relieved by the administration of luteoloside (EC50 = 0.43 mM, selection index = 5.3). Using a plaque reduction assay, we administered luteoloside at various time points and found that the compound reduced EV71 viability in RD cells rather than increasing defensive mobilization or viral absorption. Moreover, biochemical studies focused on VP1 (a key structural protein of EV71) mRNA transcript and protein levels also revealed the inhibitory effects of luteoloside on the EV71 viral yield. Finally, we performed inhibition assays using luteoloside to evaluate its effect on recombinant 3C protease activity. Our results demonstrated that luteoloside blocked 3C protease enzymatic activity in a dose-dependent manner (IC50 = 0.36 mM) that was similar to the effect of rutin, which is a well-known C3 protease inhibitor. Collectively, the results from this study indicate that luteoloside can block 3C protease activity and subsequently inhibit EV71 production in vitro. PMID:26870944

  17. Porcine Epidemic Diarrhea Virus 3C-Like Protease Regulates Its Interferon Antagonism by Cleaving NEMO

    PubMed Central

    Wang, Dang; Fang, Liurong; Shi, Yanling; Zhang, Huan; Gao, Li; Peng, Guiqing; Chen, Huanchun; Li, Kui

    2015-01-01

    ABSTRACT Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-κB activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and this cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses. IMPORTANCE The continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN

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

    PubMed

    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

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

    PubMed

    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.

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

    PubMed Central

    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

  1. Structural and Inhibitor Studies of Norovirus 3C-like Proteases

    PubMed Central

    Takahashi, Daisuke; Kim, Yunjeong; Lovell, Scott; Prakash, Om; Groutas, William C; Chang, Kyeong-Ok

    2013-01-01

    Noroviruses have a single-stranded, positive sense 7–8 kb RNA genome, which encodes a polyprotein precursor processed by a virus-encoded 3C-like cysteine protease (3CLpro) to generate mature non-structural proteins. Because processing of the polyprotein is essential for virus replication, norovirus 3CLpro has been targeted for the discovery of anti-norovirus small molecule therapeutics. Thus, we performed functional, structural and inhibition studies of norovirus 3CLpro with fluorescence resonance energy transfer (FRET) assay, X-ray crystallography, and NMR spectroscopy with a synthetic protease inhibitor. Three 3CLpro from Norwalk virus (NV, genogroup I), MD145 (genogroup II) and murine norovirus-1 (MNV-1, genogroup V) were optimized for a FRET assay, and compared for the inhibitory activities of a synthetic protease inhibitor (GC376). The apo 3D structures of NV 3CLpro determined with X-ray crystallography and NMR spectroscopy were further analyzed. In addition, the binding mode of NV 3CLpro-GC376 was compared with X-ray crystallography and NMR spectroscopy. The results of this report provide insight into the interaction of NV 3CLpro with substrate/inhibitor for better understanding of the enzyme and antiviral drug development. PMID:24055466

  2. Characterization of an Alphamesonivirus 3C-Like Protease Defines a Special Group of Nidovirus Main Proteases

    PubMed Central

    Blanck, Sandra; Stinn, Anne; Tsiklauri, Lali; Zirkel, Florian; Junglen, Sandra

    2014-01-01

    ABSTRACT Cavally virus (CavV) and related viruses in the family Mesoniviridae diverged profoundly from other nidovirus lineages but largely retained the characteristic set of replicative enzymes conserved in the Coronaviridae and Roniviridae. The expression of these enzymes in virus-infected cells requires the extensive proteolytic processing of two large replicase polyproteins, pp1a and pp1ab, by the viral 3C-like protease (3CLpro). Here, we show that CavV 3CLpro autoproteolytic cleavage occurs at two N-terminal (N1 and N2) and one C-terminal (C1) processing site(s). The mature form of 3CLpro was revealed to be a 314-residue protein produced by cleavage at FKNK1386|SAAS (N2) and YYNQ1700|SATI (C1). Site-directed mutagenesis data suggest that the mesonivirus 3CLpro employs a catalytic Cys-His dyad comprised of CavV pp1a/pp1ab residues Cys-1539 and His-1434. The study further suggests that mesonivirus 3CLpro substrate specificities differ from those of related nidovirus proteases. The presence of Gln (or Glu) at the P1 position was not required for cleavage, although residues that control Gln/Glu specificity in related viral proteases are retained in the CavV 3CLpro sequence. Asn at the P2 position was identified as a key determinant for mesonivirus 3CLpro substrate specificity. Other positions, including P4 and P1′, each are occupied by structurally related amino acids, indicating a supportive role in substrate binding. Together, the data identify a new subgroup of nidovirus main proteases and support previous conclusions on phylogenetic relationships between the main nidovirus lineages. IMPORTANCE Mesoniviruses have been suggested to provide an evolutionary link between nidovirus lineages with small (13 to 16 kb) and large (26 to 32 kb) RNA genome sizes, and it has been proposed that a specific set of enzymes, including a proofreading exoribonuclease and other replicase gene-encoded proteins, play a key role in the major genome expansion leading to the currently

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

    PubMed

    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

  4. Broad-Spectrum Inhibitors against 3C-Like Proteases of Feline Coronaviruses and Feline Caliciviruses

    PubMed Central

    Shivanna, Vinay; Narayanan, Sanjeev; Prior, Allan M.; Weerasekara, Sahani; Hua, Duy H.; Kankanamalage, Anushka C. Galasiti; Groutas, William C.; Chang, Kyeong-Ok

    2015-01-01

    ABSTRACT Feline infectious peritonitis and virulent, systemic calicivirus infection are caused by certain types of feline coronaviruses (FCoVs) and feline caliciviruses (FCVs), respectively, and are important infectious diseases with high fatality rates in members of the Felidae family. While FCoV and FCV belong to two distinct virus families, the Coronaviridae and the Caliciviridae, respectively, they share a dependence on viral 3C-like protease (3CLpro) for their replication. Since 3CLpro is functionally and structurally conserved among these viruses and essential for viral replication, 3CLpro is considered a potential target for the design of antiviral drugs with broad-spectrum activities against these distinct and highly important viral infections. However, small-molecule inhibitors against the 3CLpro enzymes of FCoV and FCV have not been previously identified. In this study, derivatives of peptidyl compounds targeting 3CLpro were synthesized and evaluated for their activities against FCoV and FCV. The structures of compounds that showed potent dual antiviral activities with a wide margin of safety were identified and are discussed. Furthermore, the in vivo efficacy of 3CLpro inhibitors was evaluated using a mouse model of coronavirus infection. Intraperitoneal administration of two 3CLpro inhibitors in mice infected with murine hepatitis virus A59, a hepatotropic coronavirus, resulted in significant reductions in virus titers and pathological lesions in the liver compared to the findings for the controls. These results suggest that the series of 3CLpro inhibitors described here may have the potential to be further developed as therapeutic agents against these important viruses in domestic and wild cats. This study provides important insights into the structure and function relationships of 3CLpro for the design of antiviral drugs with broader antiviral activities. IMPORTANCE Feline infectious peritonitis virus (FIPV) is the leading cause of death in young cats

  5. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo

    SciTech Connect

    Schlax, Peter E.; Zhang Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T. Glen . E-mail: tlawson@bates.edu

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination.

  6. Degradation of the encephalomyocarditis virus and hepatitis A virus 3C proteases by the ubiquitin/26S proteasome system in vivo.

    PubMed

    Schlax, Peter E; Zhang, Jin; Lewis, Elizabeth; Planchart, Antonio; Lawson, T Glen

    2007-04-10

    We have isolated stably transfected mouse embryonic fibroblast cell lines that inducibly express either the mature encephalomyocarditis virus (EMCV) or hepatitis A virus (HAV) 3C protease and have used these cells to demonstrate that both proteins are subject to degradation in vivo by the ubiquitin/26S proteasome system. The detection of 3C protease expression in these cells requires inducing conditions and the presence of one of several proteasome inhibitors. Both 3C proteases are incorporated into conjugates with ubiquitin in vivo. HAV 3C protease expression has deleterious effects on cell viability, as determined by observation and counting of cells cultured in the absence or presence of inducing conditions. The EMCV 3C protease was found to be preferentially localized to the nucleus of induced cells, while the HAV 3C protease remains in the cytoplasm. The absence of polyubiquitinated EMCV 3C protease conjugates in nuclear fraction preparations suggests that localization to the nucleus can protect this protein from ubiquitination. PMID:17150238

  7. Synthesis and structure-activity relationship of α-keto amides as enterovirus 71 3C protease inhibitors.

    PubMed

    Zeng, Debin; Ma, Yuying; Zhang, Rui; Nie, Quandeng; Cui, Zhengjie; Wang, Yaxin; Shang, Luqing; Yin, Zheng

    2016-04-01

    α-Keto amide derivatives as enterovirus 71 (EV71) 3C protease (3C(pro)) inhibitors have been synthesized and assayed for their biochemical and antiviral activities. structure-activity relationship (SAR) study indicated that small moieties were primarily tolerated at P1' and the introduction of para-fluoro benzyl at P2 notably improved the potency of inhibitor. Inhibitors 8v, 8w and 8x exhibited satisfactory activity (IC50=1.32±0.26μM, 1.88±0.35μM and 1.52±0.31μM, respectively) and favorable CC50 values (CC50>100μM). α-Keto amide may represent a good choice as a warhead for EV71 3C(pro) inhibitor.

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

    PubMed Central

    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

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

    PubMed

    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

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

    SciTech Connect

    Cooper, Jon; Coates, Leighton; Hussey, Robert

    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.

  11. Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

    NASA Astrophysics Data System (ADS)

    Becker, Daniel; Kaczmarska, Zuzanna; Arkona, Christoph; Schulz, Robert; Tauber, Carolin; Wolber, Gerhard; Hilgenfeld, Rolf; Coll, Miquel; Rademann, Jörg

    2016-09-01

    Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme-inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases.

  12. Irreversible inhibitors of the 3C protease of Coxsackie virus through templated assembly of protein-binding fragments

    PubMed Central

    Becker, Daniel; Kaczmarska, Zuzanna; Arkona, Christoph; Schulz, Robert; Tauber, Carolin; Wolber, Gerhard; Hilgenfeld, Rolf; Coll, Miquel; Rademann, Jörg

    2016-01-01

    Small-molecule fragments binding to biomacromolecules can be starting points for the development of drugs, but are often difficult to detect due to low affinities. Here we present a strategy that identifies protein-binding fragments through their potential to induce the target-guided formation of covalently bound, irreversible enzyme inhibitors. A protein-binding nucleophile reacts reversibly with a bis-electrophilic warhead, thereby positioning the second electrophile in close proximity of the active site of a viral protease, resulting in the covalent de-activation of the enzyme. The concept is implemented for Coxsackie virus B3 3C protease, a pharmacological target against enteroviral infections. Using an aldehyde-epoxide as bis-electrophile, active fragment combinations are validated through measuring the protein inactivation rate and by detecting covalent protein modification in mass spectrometry. The structure of one enzyme–inhibitor complex is determined by X-ray crystallography. The presented warhead activation assay provides potent non-peptidic, broad-spectrum inhibitors of enteroviral proteases. PMID:27677239

  13. Peptidyl aldehyde NK-1.8k suppresses enterovirus 71 and enterovirus 68 infection by targeting protease 3C.

    PubMed

    Wang, Yaxin; Yang, Ben; Zhai, Yangyang; Yin, Zheng; Sun, Yuna; Rao, Zihe

    2015-05-01

    Enterovirus (EV) is one of the major causative agents of hand, foot, and mouth disease in the Pacific-Asia region. In particular, EV71 causes severe central nervous system infections, and the fatality rates from EV71 infection are high. Moreover, an outbreak of respiratory illnesses caused by an emerging EV, EV68, recently occurred among over 1,000 young children in the United States and was also associated with neurological infections. Although enterovirus has emerged as a considerable global public health threat, no antiviral drug for clinical use is available. In the present work, we screened our compound library for agents targeting viral protease and identified a peptidyl aldehyde, NK-1.8k, that inhibits the proliferation of different EV71 strains and one EV68 strain and that had a 50% effective concentration of 90 nM. Low cytotoxicity (50% cytotoxic concentration, >200 μM) indicated a high selective index of over 2,000. We further characterized a single amino acid substitution inside protease 3C (3C(pro)), N69S, which conferred EV71 resistance to NK-1.8k, possibly by increasing the flexibility of the substrate binding pocket of 3C(pro). The combination of NK-1.8k and an EV71 RNA-dependent RNA polymerase inhibitor or entry inhibitor exhibited a strong synergistic anti-EV71 effect. Our findings suggest that NK-1.8k could potentially be developed for anti-EV therapy. PMID:25691647

  14. X-Ray Structure and Inhibition of 3C-like Protease from Porcine Epidemic Diarrhea Virus

    PubMed Central

    St. John, Sarah E.; Anson, Brandon J.; Mesecar, Andrew D.

    2016-01-01

    Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CLpro) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 Å X-ray structure of 3CLpro from PEDV. Analysis of the PEDV 3CLpro structure and comparison to other coronaviral 3CLpro’s from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CLpro’s are conserved, with the exception of a loop that comprises the protease S2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro, (R)-16, to have inhibitor activity against PEDV 3CLpro, despite that SARS-3CLpro and PEDV 3CLpro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CLpro are conserved in PEDV-3CLpro; however, the sequence variation and positional difference in the loop forming the S2 pocket may account for large observed difference in IC50 values. This work advances our understanding of the subtle, but important, differences in coronaviral 3CLpro architecture and contributes to the broader structural knowledge of coronaviral 3CLpro’s. PMID:27173881

  15. Structures of the Middle East respiratory syndrome coronavirus 3C-like protease reveal insights into substrate specificity

    PubMed Central

    Needle, Danielle; Lountos, George T.; Waugh, David S.

    2015-01-01

    Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic virus that causes severe respiratory illness accompanied by multi-organ dysfunction, resulting in a case fatality rate of approximately 40%. As found in other coronaviruses, the majority of the positive-stranded RNA MERS-CoV genome is translated into two polyproteins, one created by a ribosomal frameshift, that are cleaved at three sites by a papain-like protease and at 11 sites by a 3C-like protease (3CLpro). Since 3CLpro is essential for viral replication, it is a leading candidate for therapeutic intervention. To accelerate the development of 3CLpro inhibitors, three crystal structures of a catalytically inactive variant (C148A) of the MERS-CoV 3CLpro enzyme were determined. The aim was to co-crystallize the inactive enzyme with a peptide substrate. Fortuitously, however, in two of the structures the C-terminus of one protomer is bound in the active site of a neighboring molecule, providing a snapshot of an enzyme–product complex. In the third structure, two of the three protomers in the asymmetric unit form a homodimer similar to that of SARS-CoV 3CLpro; however, the third protomer adopts a radically different conformation that is likely to correspond to a crystallographic monomer, indicative of substantial structural plasticity in the enzyme. The results presented here provide a foundation for the structure-based design of small-molecule inhibitors of the MERS-CoV 3CLpro enzyme. PMID:25945576

  16. 3C-like protease of rabbit hemorrhagic disease virus: identification of cleavage sites in the ORF1 polyprotein and analysis of cleavage specificity.

    PubMed Central

    Wirblich, C; Sibilia, M; Boniotti, M B; Rossi, C; Thiel, H J; Meyers, G

    1995-01-01

    Rabbit hemorrhagic disease virus, a positive-stranded RNA virus of the family Caliciviridae, encodes a trypsin-like cysteine protease as part of a large polyprotein. Upon expression in Escherichia coli, the protease releases itself from larger precursors by proteolytic cleavages at its N and C termini. Both cleavage sites were determined by N-terminal sequence analysis of the cleavage products. Cleavage at the N terminus of the protease occurred with high efficiency at an EG dipeptide at positions 1108 and 1109. Cleavage at the C terminus of the protease occurred with low efficiency at an ET dipeptide at positions 1251 and 1252. To study the cleavage specificity of the protease, amino acid substitutions were introduced at the P2, P1, and P1' positions at the cleavage site at the N-terminal boundary of the protease. This analysis showed that the amino acid at the P1 position is the most important determinant for substrate recognition. Only glutamic acid, glutamine, and aspartic acid were tolerated at this position. At the P1' position, glycine, serine, and alanine were the preferred substrates of the protease, but a number of amino acids with larger side chains were also tolerated. Substitutions at the P2 position had only little effect on the cleavage efficiency. Cell-free expression of the C-terminal half of the ORF1 polyprotein showed that the protease catalyzes cleavage at the junction of the RNA polymerase and the capsid protein. An EG dipeptide at positions 1767 and 1768 was identified as the putative cleavage site. Our data show that rabbit hemorrhagic disease virus encodes a trypsin-like cysteine protease that is similar to 3C proteases with regard to function and specificity but is more similar to 2A proteases with regard to size. PMID:7474137

  17. X-ray structure and inhibition of 3C-like protease from porcine epidemic diarrhea virus

    DOE PAGES

    St. John, Sarah E.; Anson, Brandon J.; Mesecar, Andrew D.

    2016-05-13

    Porcine epidemic diarrhea virus (PEDV) is a coronavirus that infects pigs and can have mortality rates approaching 100% in piglets, causing serious economic impact. The 3C-like protease (3CLpro) is essential for the coronaviral life cycle and is an appealing target for the development of therapeutics. We report the expression, purification, crystallization and 2.10 angstrom X-ray structure of 3CLpro from PEDV. Analysis of the PEDV 3CLpro structure and comparison to other coronaviral 3CLpro's from the same alpha-coronavirus phylogeny shows that the overall structures and active site architectures across 3CLpro's are conserved, with the exception of a loop that comprises the proteasemore » S-2 pocket. We found a known inhibitor of severe acute respiratory syndrome coronavirus (SARS-CoV) 3CLpro, (R)-16, to have inhibitor activity against PEDV 3CLpro, despite that SARS-3CLpro and PEDV 3CLpro share only 45.4% sequence identity. Structural comparison reveals that the majority of residues involved in (R)-16 binding to SARS-3CLpro are conserved in PEDV-3CLpro; however, the sequence variation and positional difference in the loop forming the S-2 pocket may account for large observed difference in IC50 values. In conclusion, this work advances our understanding of the subtle, but important, differences in coronaviral 3CLpro architecture and contributes to the broader structural knowledge of coronaviral 3CLpro's.« less

  18. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity

    PubMed Central

    Herod, Morgan R.; Prince, Cynthia A.; Skilton, Rachel J.; Ward, Vernon K.; Cooper, Jonathan B.; Clarke, Ian N.

    2014-01-01

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

  19. Structure-based design and functional studies of novel noroviral 3C protease chimaeras offer insights into substrate specificity.

    PubMed

    Herod, Morgan R; Prince, Cynthia A; Skilton, Rachel J; Ward, Vernon K; Cooper, Jonathan B; Clarke, Ian N

    2014-12-15

    The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein. PMID:25275273

  20. Encephalomyocarditis virus 3C protease: efficient cell-free expression from clones which link viral 5' noncoding sequences to the P3 region.

    PubMed Central

    Parks, G D; Duke, G M; Palmenberg, A C

    1986-01-01

    All picornaviral peptides are derived by progressive posttranslational cleavage of a giant precursor polyprotein. Translation of encephalomyocarditis virus (EMC) RNA in rabbit reticulocyte extracts produces active viral peptides, including protease 3C, which is responsible for many cleavage reactions within the processing cascade. DNA plasmids containing 5' noncoding sequences of EMC linked to other portions of the viral genome were constructed and transcribed into RNA. Like virion RNA, the clone-derived transcripts directed efficient protein translation in vitro. The 5'-linked constructions may represent examples of a general method for cell-free expression of any cloned gene segment. One construction produced a self-cleaving P3 region precursor, which contained active 3C protease. A genetically engineered insertion within the 3C sequences eliminated endogenous self-cleavage activity without altering the ability of the P3 peptide to serve as substrate in bimolecular reactions with added 3C. Another plasmid encoding the L-VP0 portion of the capsid region was used to demonstrate that scission between the leader peptide (L) and capsid protein VP0 can be catalyzed by 3C. The enzyme responsible for this step was previously unidentified. A rapid purification scheme for isolation of 3C from EMC-infected HeLa cells is also presented. Images PMID:3021972

  1. Proteolytic processing of tomato ringspot nepovirus 3C-like protease precursors: definition of the domains for the VPg, protease and putative RNA-dependent RNA polymerase.

    PubMed

    Wang, A; Carrier, K; Chisholm, J; Wieczorek, A; Huguenot, C; Sanfaçon, H

    1999-03-01

    Tomato ringspot nepovirus (TomRSV) RNA-1 encodes a putative NTP-binding protein (NTB), a putative viral genome-linked protein (VPg), a putative RNA-dependent RNA polymerase (Pol) and a serine-like protease (Pro), which have been suggested to be involved in viral RNA replication. Proteolytic processing of protease precursors containing these proteins was studied in Escherichia coli and in vitro. The TomRSV protease could cleave the precursor proteins and release the predicted mature proteins or intermediate precursors. Although processing was detected at all three predicted cleavage sites (NTB-VPg, VPg-Pro and Pro-Pol), processing at the VPg-Pro cleavage site was inefficient, resulting in accumulation of the VPg-Pro intermediate precursor in E. coli and in vitro. In addition, the presence of the VPg sequence in the precursor resulted in increased cleavage at the Pro-Pol cleavage site in E. coli and in vitro. Direct N-terminal sequencing of the genomic RNA-linked VPg, of the mature protease purified from E. coli extracts and of radiolabelled mature polymerase purified from in vitro translation products revealed the sequences of the NTB-VPg, VPg-Pro and Pro-Pol dipeptide cleavage sites to be Q/S, Q/G and Q/S, respectively. In vitro processing at the NTB-VPg and Pro-Pol cleavage sites was not detected upon mutation or deletion of the conserved glutamine at the -1 position of the cleavage site. These results are discussed in light of the cleavage site specificity of the TomRSV protease.

  2. PARP9-DTX3L ubiquitin ligase targets host histone H2BJ and viral 3C protease to enhance interferon signaling and control viral infection

    PubMed Central

    Zhang, Yong; Mao, Dailing; Roswit, William T.; Jin, Xiaohua; Patel, Anand C.; Patel, Dhara A.; Agapov, Eugene; Wang, Zhepeng; Tidwell, Rose M.; Atkinson, Jeffrey J.; Huang, Guangming; McCarthy, Ronald; Yu, Jinsheng; Yun, Nadezhda E.; Paessler, Slobodan; Lawson, T. Glen; Omattage, Natalie S.; Brett, Tom J.; Holtzman, Michael J.

    2015-01-01

    Enhancing the response to interferon could offer an immunological advantage to the host. In support of this concept, we used a modified form of the transcription factor STAT1 to achieve interferon hyperresponsiveness without toxicity and markedly improve antiviral function in transgenic mice and transduced human cells. We found that the improvement depends on expression of a PARP9-DTX3L complex with distinct domains for interaction with STAT1 and for activity as an E3 ubiquitin ligase that acts on host histone H2BJ to promote interferon-stimulated gene expression and on viral 3C proteases to initiate their degradation via the immunoproteasome. Together, PARP9-DTX3L acts on host and pathogen to achieve a double layer of immunity within a safe reserve in the interferon signaling pathway. PMID:26479788

  3. 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.

    PubMed

    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.

  4. Protection of guinea pigs and swine by a recombinant adenovirus expressing O serotype of foot-and-mouth disease virus whole capsid and 3C protease.

    PubMed

    Lu, Zengjun; Bao, Huifang; Cao, Yimei; Sun, Pu; Guo, Jianhun; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Li, Dong; Liu, Zaixin; Xie, Qingge

    2008-12-19

    Two recombinant adenoviruses were constructed expressing foot-and-mouth disease virus (FMDV) capsid and 3C/3CD proteins in replicative deficient human adenovirus type 5 vector. Guinea pigs vaccinated with 1-3 x 10(8)TCID(50) Ad-P12x3C recombinant adenovirus were completely protected against 10,000GID(50) homologous virulent FMDV challenge 25 days post vaccination (dpv). Ad-P12x3CD vaccinated guinea pigs were only partially protected. Swine were vaccinated once with 1x10(9)TCID(50) Ad-P12x3C hybrid virus and challenged 28 days later. Three of four vaccinated swine were completely protected against 200 pig 50% infectious doses (ID(50)) of homologous FMDV challenge, and vaccinated pigs developed specific cellular and humoral immune responses. The immune effect of Ad-P12x3C in swine further indicated that the recombinant adenovirus was highly efficient in transferring the foreign gene. This approach may thus be a very hopeful tool for developing FMD live virus vector vaccine. PMID:19178894

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

    SciTech Connect

    Lawrence, Paul; Schafer, Elizabeth A.; Rieder, Elizabeth

    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}.

  6. Multiple Poliovirus Proteins Repress Cytoplasmic RNA Granules

    PubMed Central

    Dougherty, Jonathan D.; Tsai, Wei-Chih; Lloyd, Richard E.

    2015-01-01

    We have previously shown that poliovirus (PV) infection induces stress granule (SG) formation early in infection and then inhibits the formation of SG and disperses processing bodies (PBs) by the mid-phase of infection. Loss of SG was linked to cleavage of G3BP1 by viral 3C proteinase (3Cpro), however dispersal of PBs was not strongly linked to cleavage of specific factors by viral proteinases, suggesting other viral proteins may play roles in inhibition of SG or PB formation. Here we have screened all viral proteins for roles in inducing or inhibiting the formation of RNA granules by creating fusions with mCherry and expressing them individually in cells. Expression of viral proteins separately revealed that the capsid region P1, 2Apro, 3A, 3Cpro, the protease precursor 3CD and 3D polymerase all affect RNA granules to varying extents, whereas 2BC does not. 2Apro, which cleaves eIF4GI, induced SGs as expected, and entered novel foci containing the SG nucleating protein G3BP1. Of the two forms of G3BP, only G3BP1 is cleaved by a virus proteinase, 3Cpro, whereas G3BP2 is not cleaved by 3Cpro or 2Apro. Surprisingly, 3CD, which contains proteinase activity, differentially repressed PBs but not SGs. Further, both 2Apro and 3Cpro expression dispersed PBs, however molecular targets were different since PB dispersal due to 2Apro and heat shock protein (Hsp)90 inhibition but not 3Cpro, could be rescued by application of oxidative stress to cells. The data indicate that PV repression of SGs and PBs is multifactorial, though protease function is dominant. PMID:26610553

  7. Supermarket Proteases.

    ERIC Educational Resources Information Center

    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)

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

    SciTech Connect

    Perley, R. A.; Butler, B. J. E-mail: BButler@nrao.edu

    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.

  9. Proteases as Insecticidal Agents

    PubMed Central

    Harrison, Robert L.; Bonning, Bryony C.

    2010-01-01

    Proteases from a variety of sources (viruses, bacteria, fungi, plants, and insects) have toxicity towards insects. Some of these insecticidal proteases evolved as venom components, herbivore resistance factors, or microbial pathogenicity factors, while other proteases play roles in insect development or digestion, but exert an insecticidal effect when over-expressed from genetically engineered plants or microbial pathogens. Many of these proteases are cysteine proteases, although insect-toxic metalloproteases and serine proteases have also been examined. The sites of protease toxic activity range from the insect midgut to the hemocoel (body cavity) to the cuticle. This review discusses these insecticidal proteases along with their evaluation and use as potential pesticides. PMID:22069618

  10. Investigations with Protease.

    ERIC Educational Resources Information Center

    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)

  11. Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14

    NASA Astrophysics Data System (ADS)

    Leong, L. E.-C.; Walker, P. A.; Porter, A. G.

    1992-08-01

    The protease (3C pro) from human rhinovirus serotype-14 (HRV-14) has been cloned and efficiently expressed in E. coli. A straightforward single-step purification of the recombinant 3C pro has been achieved by fusing the protein to the car☐y-terminus of the glutathione-S-transferase from Schistosoma japonicum. Modifications made to the 5' end of the PCR fragment coding for the 3C pro have allowed the specific cleavage of the fusion protein using thrombin to yield mature 3C pro with the correct amino-terminal amino acid. This protease has been shown to be active when assayed using synthetic peptides corresponding to the natural cleavage recognition sequences within the polyprotein. Other substrates are being developed for this protease for possible use in the screening of inhibitors of 3C pro. Sufficient protease 3C pro has been purified for initial attempts at crystallization.

  12. Proteases of human rhinovirus: role in infection.

    PubMed

    Jensen, Lora M; Walker, Erin J; Jans, David A; Ghildyal, Reena

    2015-01-01

    Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2A(pro) and 3C(pro), the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2A(pro) and 3C(pro) are clearly viable drug targets, and indeed, 3C(pro) has been successfully targeted for treating the common cold in experimental infection. 2A(pro) and 3C(pro) are crucial for virus replication due to their role in polyprotein processing as well as cleavage of key cellular proteins to inhibit cellular transcription and translation. Intriguingly, the action of the HRV proteases also disrupts nucleocytoplasmic trafficking, contributing to HRV cytopathic effects. Improved understanding of the protease-cell interactions should enable new therapeutic approaches to be identified for drug development. PMID:25261311

  13. Proteases of human rhinovirus: role in infection.

    PubMed

    Jensen, Lora M; Walker, Erin J; Jans, David A; Ghildyal, Reena

    2015-01-01

    Human rhinoviruses (HRV) are the major etiological agents of the common cold and asthma exacerbations, with significant worldwide health and economic impact. Although large-scale population vaccination has proved successful in limiting or even eradicating many viruses, the more than 100 distinct serotypes mean that conventional vaccination is not a feasible strategy to combat HRV. An alternative strategy is to target conserved viral proteins such as the HRV proteases, 2A(pro) and 3C(pro), the focus of this review. Necessary for host cell shutoff, virus replication, and pathogenesis, 2A(pro) and 3C(pro) are clearly viable drug targets, and indeed, 3C(pro) has been successfully targeted for treating the common cold in experimental infection. 2A(pro) and 3C(pro) are crucial for virus replication due to their role in polyprotein processing as well as cleavage of key cellular proteins to inhibit cellular transcription and translation. Intriguingly, the action of the HRV proteases also disrupts nucleocytoplasmic trafficking, contributing to HRV cytopathic effects. Improved understanding of the protease-cell interactions should enable new therapeutic approaches to be identified for drug development.

  14. Proteases as therapeutics

    PubMed Central

    Craik, Charles S.; Page, Michael J.; Madison, Edwin L.

    2015-01-01

    Proteases are an expanding class of drugs that hold great promise. The U.S. FDA (Food and Drug Administration) has approved 12 protease therapies, and a number of next generation or completely new proteases are in clinical development. Although they are a well-recognized class of targets for inhibitors, proteases themselves have not typically been considered as a drug class despite their application in the clinic over the last several decades; initially as plasma fractions and later as purified products. Although the predominant use of proteases has been in treating cardiovascular disease, they are also emerging as useful agents in the treatment of sepsis, digestive disorders, inflammation, cystic fibrosis, retinal disorders, psoriasis and other diseases. In the present review, we outline the history of proteases as therapeutics, provide an overview of their current clinical application, and describe several approaches to improve and expand their clinical application. Undoubtedly, our ability to harness proteolysis for disease treatment will increase with our understanding of protease biology and the molecular mechanisms responsible. New technologies for rationally engineering proteases, as well as improved delivery options, will expand greatly the potential applications of these enzymes. The recognition that proteases are, in fact, an established class of safe and efficacious drugs will stimulate investigation of additional therapeutic applications for these enzymes. Proteases therefore have a bright future as a distinct therapeutic class with diverse clinical applications. PMID:21406063

  15. 18 CFR 3c.2 - Nonpublic information.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 18 Conservation of Power and Water Resources 1 2010-04-01 2010-04-01 false Nonpublic information. 3c.2 Section 3c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES STANDARDS OF CONDUCT § 3c.2 Nonpublic information. (a) Section 1264(d)...

  16. 18 CFR 3c.2 - Nonpublic information.

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 18 Conservation of Power and Water Resources 1 2014-04-01 2014-04-01 false Nonpublic information. 3c.2 Section 3c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES STANDARDS OF CONDUCT § 3c.2 Nonpublic information. (a) Section 1264(d)...

  17. 18 CFR 3c.2 - Nonpublic information.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 18 Conservation of Power and Water Resources 1 2011-04-01 2011-04-01 false Nonpublic information. 3c.2 Section 3c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES STANDARDS OF CONDUCT § 3c.2 Nonpublic information. (a) Section 1264(d)...

  18. 18 CFR 3c.2 - Nonpublic information.

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 18 Conservation of Power and Water Resources 1 2012-04-01 2012-04-01 false Nonpublic information. 3c.2 Section 3c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES STANDARDS OF CONDUCT § 3c.2 Nonpublic information. (a) Section 1264(d)...

  19. 18 CFR 3c.2 - Nonpublic information.

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 18 Conservation of Power and Water Resources 1 2013-04-01 2013-04-01 false Nonpublic information. 3c.2 Section 3c.2 Conservation of Power and Water Resources FEDERAL ENERGY REGULATORY COMMISSION, DEPARTMENT OF ENERGY GENERAL RULES STANDARDS OF CONDUCT § 3c.2 Nonpublic information. (a) Section 1264(d)...

  20. Simple in vitro translation assay to analyze inhibitors of rhinovirus proteases.

    PubMed Central

    Heinz, B A; Tang, J; Labus, J M; Chadwell, F W; Kaldor, S W; Hammond, M

    1996-01-01

    We have developed a simple in vitro translation method to analyze compounds that inhibit the rhinovirus 3C protease in peptide substrate assays but demonstrate no antiviral activity. This complementary assay, which provides both qualitative and quantitative results, detects the inhibition of the 3CD protease in the native polyprotein form. PMID:8787922

  1. Viral cysteine proteases are homologous to the trypsin-like family of serine proteases: structural and functional implications.

    PubMed Central

    Bazan, J F; Fletterick, R J

    1988-01-01

    Proteases that are encoded by animal picornaviruses and plant como- and potyviruses form a related group of cysteine-active-center enzymes that are essential for virus maturation. We show that these proteins are homologous to the family of trypsin-like serine proteases. In our model, the active-site nucleophile of the trypsin catalytic triad, Ser-195, is changed to a Cys residue in these viral proteases. The other two residues of the triad, His-57 and Asp-102, are otherwise absolutely conserved in all the viral protease sequences. Secondary structure analysis of aligned sequences suggests the location of the component strands of the twin beta-barrel trypsin fold in the viral proteases. Unexpectedly, the 2a and 3c subclasses of viral cysteine proteases are, respectively, homologous to the small and large structural subclasses of trypsin-like serine proteases. This classification allows the molecular mapping of residues from viral sequences onto related tertiary structures; we precisely identify amino acids that are strong determinants of specificity for both small and large viral cysteine proteases. Images PMID:3186696

  2. The high energy source 3C 273

    NASA Technical Reports Server (NTRS)

    Vonmontigny, Corinna

    1990-01-01

    The properties of 3C 273 are reviewed and an attempt is made to find an answer to the question why 3C 273 is the only extragalactic source so far, which was detected at energies greater than or equal to 50 MeV.

  3. NATO-3C/Delta launch

    NASA Technical Reports Server (NTRS)

    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.

  4. The Lon AAA+ protease.

    PubMed

    Gur, Eyal

    2013-01-01

    As the first ATP-dependent protease to be identified, Lon holds a special place in the history of cellular biology. In fact, the concept of ATP-dependent protein degradation was established through the findings that led to the discovery of Lon. Therefore, this chapter begins with a historical perspective, describing the milestones that led to the discovery of Lon and ATP-dependent proteolysis, starting from the early findings in the 1960s until the demonstration of Lon's ATP-dependent proteolytic activity in vitro, in 1981. Most of our knowledge on Lon derives from studies of the Escherichia coli Lon ortholog, and, therefore, most of this chapter relates to this particular enzyme. Nonetheless, Lon is not only found in most bacterial species, it is also found in Archaea and in the mitochondrion and chloroplast of eukaryotic cells. Therefore many of the conclusions gained from studies on the E. coli enzyme are relevant to Lon proteases in other organisms. Lon, more than any other bacterial or organellar protease, is associated with the degradation of misfolded proteins and protein quality control. In addition, Lon also degrades many regulatory proteins that are natively folded, thus it also plays a prominent role in regulation of physiological processes. Throughout the years, many Lon substrates have been identified, confirming its role in the regulation of diverse cellular processes, including cell division, DNA replication, differentiation, and adaptation to stress conditions. Some examples of these functions are described and discussed here, as is the role of Lon in the degradation of misfolded proteins and in protein quality control. Finally, this chapter deals with the exquisite sensitivity of protein degradation inside a cell. How can a protease distinguish so many substrates from cellular proteins that should not be degraded? Can the specificity of a protease be regulated according to the physiological needs of a cell? This chapter thus broadly discusses the

  5. Laundry performance of subtilisin proteases.

    PubMed

    Wolff, A M; Showell, M S; Venegas, M G; Barnett, B L; Wertz, W C

    1996-01-01

    Effective laundry protease performance against susceptible stains depends upon both the enzyme itself and the environment in which it must work. In order to technically design superior laundry proteases, a model for protease's mechanism of action in detergents was developed which has been substantiated through-the-wash. While evaluation of this model and/or a given protease's effectiveness could be judged by a variety of methods, the utility of using visual wash performance comparisons, analytical, and stain characterization studies is described. Finally, data comparing the performance of wild type Subtilisin proteases with mutants designed via the projected model are given, demonstrating possible utility of the system.

  6. From proteases to proteomics.

    PubMed

    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).

  7. From proteases to proteomics

    PubMed Central

    Neurath, Hans

    2001-01-01

    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). PMID:11274481

  8. Protease signalling: the cutting edge

    PubMed Central

    Turk, Boris; Turk, Dus̆an; Turk, Vito

    2012-01-01

    Protease research has undergone a major expansion in the last decade, largely due to the extremely rapid development of new technologies, such as quantitative proteomics and in-vivo imaging, as well as an extensive use of in-vivo models. These have led to identification of physiological substrates and resulted in a paradigm shift from the concept of proteases as protein-degrading enzymes to proteases as key signalling molecules. However, we are still at the beginning of an understanding of protease signalling pathways. We have only identified a minor subset of true physiological substrates for a limited number of proteases, and their physiological regulation is still not well understood. Similarly, links with other signalling systems are not well established. Herein, we will highlight current challenges in protease research. PMID:22367392

  9. Protease-mediated drug delivery

    NASA Astrophysics Data System (ADS)

    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. A tunable, modular approach to fluorescent protease-activated reporters.

    PubMed

    Wu, Peng; Nicholls, Samantha B; Hardy, Jeanne A

    2013-04-01

    Proteases are one of the most important and historically utilized classes of drug targets. To effectively interrogate this class of proteins, which encodes nearly 2% of the human proteome, it is necessary to develop effective and cost-efficient methods that report on their activity both in vitro and in vivo. We have developed a robust reporter of caspase proteolytic activity, called caspase-activatable green fluorescent protein (CA-GFP). The caspases play central roles in homeostatic regulation, as they execute programmed cell death, and in drug design, as caspases are involved in diseases ranging from cancer to neurodegeneration. CA-GFP is a genetically encoded dark-to-bright fluorescent reporter of caspase activity in in vitro, cell-based, and animal systems. Based on the CA-GFP platform, we developed reporters that can discriminate the activities of caspase-6 and -7, two highly related proteases. A second series of reporters, activated by human rhinovirus 3C protease, demonstrated that we could alter the specificity of the reporter by reengineering the protease recognition sequence. Finally, we took advantage of the spectrum of known fluorescent proteins to generate green, yellow, cyan, and red reporters, paving the way for multiplex protease monitoring. PMID:23561537

  11. Protease degradable electrospun fibrous hydrogels

    PubMed Central

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

    2015-01-01

    Electrospun nanofibers 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 (MMPs). 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 fiber populations support selective fiber degradation based on individual fiber degradability. Overall, this represents a novel biomimetic approach to generate protease-sensitive fibrous scaffolds for biomedical applications. PMID:25799370

  12. Multiwavelength observations of giant radio galaxy 3C 35 and 3C 284

    NASA Astrophysics Data System (ADS)

    Pal, Sabyasachi; Chakrabarti, Sandip Kumar; Patra, Dusmanta; Konar, Chiranjib

    2016-07-01

    We report multi wavelength observations of large radio galaxy 3C35 and 3C284. The low frequency observations were done with the Giant Metrewave Radio Telescope (GMRT) starting from 150 MHz. The high frequency observations were done with Jansky Very Large Array (JVLA). Our main motivation for these observations is to estimate the spectral ages of these galaxies and to examine any proof of extended emission at low radio frequencies due to an earlier cycle of activity. The spectral age is measured by fitting the spectra with different spectral ageing models e.g. Kardashev-Pacholczyk (KP), Jaffe-Perola (JP) and Continuous Injection (CI).

  13. Multifrequency Monitoring of 3C 120, 3C 279, and PKS 1510--089

    NASA Technical Reports Server (NTRS)

    Jorstad, S. G.; Marscher, A. P.; Aller, M. F.; Balonek, T. J.; Gomez, J.-L.; McHardy, I. M.; Terasranta, H.; Raiteri, C.; Tosti, G.

    2001-01-01

    We analyze contemporaneous X-ray, optical, and radio light curves of 3C 120, ABC 279, and PKS 1510-089 on timescales from a few to hundreds of days over a 3-5 year period. The results show the diverse connections between variability properties at different frequencies for different blazers.

  14. The resolved outflow from 3C 48

    SciTech Connect

    Shih, Hsin-Yi; Stockton, Alan E-mail: stockton@ifa.hawaii.edu

    2014-10-20

    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 = 10{sup 3}-10{sup 4} cm{sup –3}, the mass is ∼6 × 10{sup 6} M {sub ☉}, 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.

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

    PubMed

    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.

  16. Serine proteases of parasitic helminths.

    PubMed

    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

  17. Serine Proteases of Parasitic Helminths

    PubMed Central

    Yang, Yong; 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 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

  18. Comparative genomics of mycobacterial proteases.

    PubMed

    Ribeiro-Guimarães, Michelle Lopes; Pessolani, Maria Cristina Vidal

    2007-01-01

    Although proteases are recognized as important virulent factors in pathogenic microorganisms, little information is available so far regarding the potential role of these enzymes in diseases caused by mycobacteria. Here we use bioinformatic tools to compare the protease-coding genes present in the genome of Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium bovis and Mycobacterium avium paratuberculosis. This analysis allowed a review of the nomenclature of the protease family present in mycobacteria. A special attention was devoted to the 'decaying genome' of M. leprae where a relatively high level of conservation of protease-coding genes was observed when compared to other genes families. A total of 39 genes out of the 49 found in M. bovis were identified in M. leprae. Of relevance, a core of well-conserved 38 protease genes shared by the four species was defined. This set of proteases is probably essential for survival in the host and disease outcome and may constitute novel targets for drug development leading to a more effective control of mycobacterial diseases.

  19. THE ACCELERATING JET OF 3C 279

    SciTech Connect

    Bloom, S. D.; Fromm, C. M.; Ros, E.

    2013-01-01

    Analysis of the proper motions of the subparsec scale jet of the quasar 3C 279 at 15 GHz with the Very Long Baseline Array shows significant accelerations in four of nine superluminal features. Analysis of these motions is combined with the analysis of flux density light curves to constrain values of Lorentz factor and viewing angle (and their derivatives) for each component. The data for each of these components are consistent with significant changes to the Lorentz factor, viewing angle, and azimuthal angle, suggesting jet bending with changes in speed. We see that for these observed components Lorentz factors are in the range {Gamma} = 10-41, viewing angles are in the range thetav = 0. Degree-Sign 1-5. Degree-Sign 0, and intrinsic (source frame) flux density is in the range, F{sub {nu},int} 1.5 Multiplication-Sign 10{sup -9}-1.5 Multiplication-Sign 10{sup -5} Jy. Considering individual components, the Lorentz factors vary from {Gamma} = 11-16 for C1, {Gamma} = 31-41 for C5, {Gamma} = 29-41 for C6, and {Gamma} = 9-12 for C8, indicating that there is no single underlying flow speed to the jet and likely we are seeing pattern speeds from shocks in the jet. The viewing angles vary in time from 0. Degree-Sign 6 to 1. Degree-Sign 5 in the case of C1 (the least extreme example), from 0. Degree-Sign 5 to 5. Degree-Sign 0 in the case of C8, and from 0. Degree-Sign 1 to 0. Degree-Sign 9 for C5 (the last two being the most extreme examples). The intrinsic flux density varies by factors from 1.4 for C8 and 430 for C5. Theoretical analysis of the accelerations also indicates potential jet bending. In addition, for one component, C5, polarization measurements also set limits to the trajectory of the jet.

  20. The Resolved Outflow from 3C 48

    NASA Astrophysics Data System (ADS)

    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

  1. RNA nuclear export is blocked by poliovirus 2A protease and is concomitant with nucleoporin cleavage.

    PubMed

    Castelló, Alfredo; Izquierdo, José M; Welnowska, Ewelina; Carrasco, Luis

    2009-10-15

    Cytopathic viruses have developed successful strategies to block or, at least, to attenuate host interference with their replication. Here, we have analyzed the effects of poliovirus 2A protease on RNA nuclear export. 2A protease interferes with trafficking of mRNAs, rRNAs and U snRNAs from the nucleus to the cytoplasm, without any apparent effect on tRNA transport. Traffic of newly produced mRNAs is more strongly affected than traffic of other mRNAs over-represented in the cytoplasm, such as mRNA encoding beta-actin. Inhibition of RNA nuclear export in HeLa cells expressing 2A protease is concomitant with the cleavage of Nup98, Nup153, Nup62 and their subsequent subcellular redistribution. The expression of an inactive 2A protease failed to interfere with RNA nuclear export. In addition, other related proteases, such as poliovirus 3C or foot and mouth disease virus L(pro) did not affect mRNA distribution or Nup98 integrity. Treatment of HeLa cells with interferon (IFN)-gamma increased the relative amount of Nup98. Under such conditions, the cleavage of Nup98 induced by 2A protease is partial, and thus IFN-gamma prevents the inhibition of RNA nuclear export. Taken together, these results are consistent with a specific proteolysis of Nup98 by 2A protease to prevent de novo mRNA traffic in poliovirus-infected cells.

  2. Peptidomimetic inhibitors of HIV protease.

    PubMed

    Randolph, John T; DeGoey, David A

    2004-01-01

    There are currently (July, 2002) six protease inhibitors approved for the treatment of HIV infection, each of which can be classified as peptidomimetic in structure. These agents, when used in combination with other antiretroviral agents, produce a sustained decrease in viral load, often to levels below the limits of quantifiable detection, and a significant reconstitution of the immune system. Therapeutic regimens containing one or more HIV protease inhibitors thus provide a highly effective method for disease management. The important role of protease inhibitors in HIV therapy, combined with numerous challenges remaining in HIV treatment, have resulted in a continued effort both to optimize regimens using the existing agents and to identify new protease inhibitors that may provide unique properties. This review will provide an overview of the discovery and clinical trials of the currently approved HIV protease inhibitors, followed by an examination of important aspects of therapy, such as pharmacokinetic enhancement, resistance and side effects. A description of new peptidomimetic compounds currently being investigated in the clinic and in preclinical discovery will follow. PMID:15193140

  3. Microbial inhibitors of cysteine proteases.

    PubMed

    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

  4. Microbial inhibitors of cysteine proteases.

    PubMed

    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.

  5. Subfamily-Specific Fluorescent Probes for Cysteine Proteases Display Dynamic Protease Activities during Seed Germination1

    PubMed Central

    Lu, Haibin; Chandrasekar, Balakumaran; Oeljeklaus, Julian; Misas-Villamil, Johana C.; Wang, Zheming; Shindo, Takayuki; Bogyo, Matthew; Kaiser, Markus; van der Hoorn, Renier A.L.

    2015-01-01

    Cysteine proteases are an important class of enzymes implicated in both developmental and defense-related programmed cell death and other biological processes in plants. Because there are dozens of cysteine proteases that are posttranslationally regulated by processing, environmental conditions, and inhibitors, new methodologies are required to study these pivotal enzymes individually. Here, we introduce fluorescence activity-based probes that specifically target three distinct cysteine protease subfamilies: aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes. We applied protease activity profiling with these new probes on Arabidopsis (Arabidopsis thaliana) protease knockout lines and agroinfiltrated leaves to identify the probe targets and on other plant species to demonstrate their broad applicability. These probes revealed that most commercially available protease inhibitors target unexpected proteases in plants. When applied on germinating seeds, these probes reveal dynamic activities of aleurain-like proteases, cathepsin B-like proteases, and vacuolar processing enzymes, coinciding with the remobilization of seed storage proteins. PMID:26048883

  6. Glycine decarboxylase in C3, C4 and C3-C4 intermediate species.

    PubMed

    Schulze, Stefanie; Westhoff, Peter; Gowik, Udo

    2016-06-01

    The glycine decarboxylase complex (GDC) plays a central role in photorespiration. GDC is localized in the mitochondria and together with serine hydroxymethyltransferase it converts two molecules of glycine to one molecule of serine, CO2 and NH3. Overexpression of GDC subunits in the C3 species Arabidopsis thaliana can increase the metabolic flux through the photorespiratory pathway leading to enhanced photosynthetic efficiency and consequently to an enhanced biomass production of the transgenic plants. Changing the spatial expression patterns of GDC subunits was an important step during the evolution of C3-C4 intermediate and likely also C4 plants. Restriction of the GDC activity to the bundle sheath cells led to the establishment of a photorespiratory CO2 pump. PMID:27038285

  7. Curcumin derivatives as HIV-1 protease inhibitors

    SciTech Connect

    Sui, Z.; Li, J.; Craik, C.S.; Ortiz de Montellano, P.R.

    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.

  8. Proteases in Fas-mediated apoptosis.

    PubMed

    Zhivotovsky, B; Burgess, D H; Schlegel, J; Pörn, M I; Vanags, D; Orrenius, S

    1997-01-01

    Involvement of a unique family of cysteine proteases in the multistep apoptotic process has been documented. Cloning of several mammalian genes identifies some components of this cellular response. However, it is currently unclear which protease plays a role as a signal and/or effector of apoptosis. We summarize contributions to the data concerning proteases in Fas-mediated apoptosis.

  9. On the Y-chromosome haplogroup C3c classification.

    PubMed

    Malyarchuk, Boris A; Derenko, Miroslava; Denisova, Galina

    2012-10-01

    As there are ambiguities in classification of the Y-chromosome haplogroup C3c, relatively frequent in populations of Northern Asia, we analyzed all three haplogroup-defining markers M48, M77 and M86 in C3-M217-individuals from Siberia, Eastern Asia and Eastern Europe. We have found that haplogroup C3c is characterized by the derived state at M48, whereas mutations at both M77 and M86 define subhaplogroup C3c1. The branch defined by M48 alone would belong to subhaplogroup C3c*, characteristic for some populations of Central and Eastern Siberia, such as Koryaks, Evens, Evenks and Yukaghirs. Subhaplogroup C3c* individuals could be considered as remnants of the Neolithic population of Siberia, based on the age of C3c*-short tandem repeat variation amounting to 4.5 ± 2.4 thousand years. PMID:22810113

  10. A Fragment-Based Method to Discover Irreversible Covalent Inhibitors of Cysteine Proteases

    PubMed Central

    2015-01-01

    A novel fragment-based drug discovery approach is reported which irreversibly tethers drug-like fragments to catalytic cysteines. We attached an electrophile to 100 fragments without significant alterations in the reactivity of the electrophile. A mass spectrometry assay discovered three nonpeptidic inhibitors of the cysteine protease papain. The identified compounds display the characteristics of irreversible inhibitors. The irreversible tethering system also displays specificity: the three identified papain inhibitors did not covalently react with UbcH7, USP08, or GST-tagged human rhinovirus 3C protease. PMID:24870364

  11. Exogenous proteases for meat tenderization.

    PubMed

    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

  12. Network Analyses Reveal Pervasive Functional Regulation Between Proteases in the Human Protease Web

    PubMed Central

    Fortelny, Nikolaus; Cox, Jennifer H.; Kappelhoff, Reinhild; Starr, Amanda E.; Lange, Philipp F.; Pavlidis, Paul; Overall, Christopher M.

    2014-01-01

    Proteolytic processing is an irreversible posttranslational modification affecting a large portion of the proteome. Protease-cleaved mediators frequently exhibit altered activity, and biological pathways are often regulated by proteolytic processing. Many of these mechanisms have not been appreciated as being protease-dependent, and the potential in unraveling a complex new dimension of biological control is increasingly recognized. Proteases are currently believed to act individually or in isolated cascades. However, conclusive but scattered biochemical evidence indicates broader regulation of proteases by protease and inhibitor interactions. Therefore, to systematically study such interactions, we assembled curated protease cleavage and inhibition data into a global, computational representation, termed the protease web. This revealed that proteases pervasively influence the activity of other proteases directly or by cleaving intermediate proteases or protease inhibitors. The protease web spans four classes of proteases and inhibitors and so links both recently and classically described protease groups and cascades, which can no longer be viewed as operating in isolation in vivo. We demonstrated that this observation, termed reachability, is robust to alterations in the data and will only increase in the future as additional data are added. We further show how subnetworks of the web are operational in 23 different tissues reflecting different phenotypes. We applied our network to develop novel insights into biologically relevant protease interactions using cell-specific proteases of the polymorphonuclear leukocyte as a system. Predictions from the protease web on the activity of matrix metalloproteinase 8 (MMP8) and neutrophil elastase being linked by an inactivating cleavage of serpinA1 by MMP8 were validated and explain perplexing Mmp8 −/− versus wild-type polymorphonuclear chemokine cleavages in vivo. Our findings supply systematically derived and

  13. Biotechnology of Cold-Active Proteases

    PubMed Central

    Joshi, Swati; Satyanarayana, Tulasi

    2013-01-01

    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. PMID:24832807

  14. Why Are C3-C4 Intermediate Species Rare?

    NASA Astrophysics Data System (ADS)

    Johnson, J. E.; Field, C. B.; Berry, J. A.

    2014-12-01

    While C3-C4 intermediate photosynthesis is thought to represent the evolutionary bridge between C3 and C4 photosynthesis, C3-C4 intermediate species are ecologically rare in comparison to both C3 and C4 species. Here, we report results from a laboratory experiment, field observations, and model simulations that suggest a new explanation for the ecological rarity of C3-C4 intermediate species. In the laboratory experiment, we combined gas exchange and fluorescence to characterize the temperature response of photosynthesis in three closely-related species in the genus Flaveria that are representatives of the C3, C3-C4 intermediate, and C4 photosynthetic pathways. The leaf temperature that maximized the quantum yield for CO2 assimilation (Topt(ΦCO2)) was 24.9 ± 0.7°C in Flaveria robusta (C3), 29.8 ± 1.0°C in F. chloraefolia (C3-C4), and 35.7 ± 0.8°C in F. bidentis (C4), and was linearly related to the temperature sensitivity of the coupling between CO2 assimilation and electron transport (d(ΦCO2/ ΦPSII)/dT)). While F. chloraefolia does not simultaneously occur with F. robusta and F. bidentis in naturally-assembled communities, this C3-C4 intermediate species does occur with other C3 and C4 species. During the growing season in two of these mixed-photosynthetic-type communities, leaf temperatures for F. chloraefolia were similar to the Topt(ΦCO2) determined in the laboratory. A model of maximum potential carbon gain suggests that competitive coexistence of C3, C3-C4 intermediate, and C4 species could be dependent on a temperature regime that highlights the distinct relative advantages of the C3-C4 intermediate pathway. In combination, these results suggest that the relative temperature sensitivity of the C3, C3-C4 intermediate, and C4 photosynthetic pathways combined with environmental variation in temperature may help to explain why C3-C4 intermediate species are generally rare.

  15. Type II Transmembrane Serine Proteases*

    PubMed Central

    Bugge, Thomas H.; Antalis, Toni M.; Wu, Qingyu

    2009-01-01

    Analysis of genome and expressed sequence tag data bases at the turn of the millennium unveiled a new protease family named the type II transmembrane serine proteases (TTSPs) in a Journal of Biological Chemistry minireview (Hooper, J. D., Clements, J. A., Quigley, J. P., and Antalis, T. M. (2001) J. Biol. Chem. 276, 857–860). Since then, the number of known TTSPs has more than doubled, and more importantly, our understanding of the physiological functions of individual TTSPs and their contribution to human disease has greatly increased. Progress has also been made in identifying molecular substrates and endogenous inhibitors. This minireview summarizes the current knowledge of the rapidly advancing TTSP field. PMID:19487698

  16. Molecular Imaging of Proteases in Cancer

    PubMed Central

    Yang, Yunan; Hong, Hao; Zhang, Yin; Cai, Weibo

    2010-01-01

    Proteases play important roles during tumor angiogenesis, invasion, and metastasis. Various molecular imaging techniques have been employed for protease imaging: optical (both fluorescence and bioluminescence), magnetic resonance imaging (MRI), single-photon emission computed tomography (SPECT), and positron emission tomography (PET). In this review, we will summarize the current status of imaging proteases in cancer with these techniques. Optical imaging of proteases, in particular with fluorescence, is the most intensively validated and many of the imaging probes are already commercially available. It is generally agreed that the use of activatable probes is the most accurate and appropriate means for measuring protease activity. Molecular imaging of proteases with other techniques (i.e. MRI, SPECT, and PET) has not been well-documented in the literature which certainly deserves much future effort. Optical imaging and molecular MRI of protease activity has very limited potential for clinical investigation. PET/SPECT imaging is suitable for clinical investigation; however the optimal probes for PET/SPECT imaging of proteases in cancer have yet to be developed. Successful development of protease imaging probes with optimal in vivo stability, tumor targeting efficacy, and desirable pharmacokinetics for clinical translation will eventually improve cancer patient management. Not limited to cancer, these protease-targeted imaging probes will also have broad applications in other diseases such as arthritis, atherosclerosis, and myocardial infarction. PMID:20234801

  17. Towards Large Area Growth of 3C-SiC

    SciTech Connect

    Vasiliauskas, Remigijus; Liljedahl, Rickard; Syvaejaervi, Mikael; Yakimova, Rositza

    2010-11-01

    In this work we have analyzed the possibility of upscaling the growth of 3C-SiC. The growth was done at different temperatures to find limiting mechanisms of the growth rate and to examine the morphology of grown layers. Coverage by 3C-SiC increases when increasing temperature, however more twins appeared. Activation energy of the growth is 130 kcal/mol--showing that growth rate limiting mechanism is sublimation of the source. We discuss the influence of large area 6H-SiC wafers on the formation of 3C-SiC, in which the change in basal plane orientation could also influence the growth of 3C-SiC.

  18. Advances in protease engineering for laundry detergents.

    PubMed

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

    2015-12-25

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

  19. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, David B.; Lao, Guifang

    1998-01-01

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

  20. Nucleotide sequences encoding a thermostable alkaline protease

    DOEpatents

    Wilson, D.B.; Lao, G.

    1998-01-06

    Nucleotide sequences, derived from a thermophilic actinomycete microorganism, which encode a thermostable alkaline protease are disclosed. Also disclosed are variants of the nucleotide sequences which encode a polypeptide having thermostable alkaline proteolytic activity. Recombinant thermostable alkaline protease or recombinant polypeptide may be obtained by culturing in a medium a host cell genetically engineered to contain and express a nucleotide sequence according to the present invention, and recovering the recombinant thermostable alkaline protease or recombinant polypeptide from the culture medium. 3 figs.

  1. Crystal structures of Bacillus subtilis Lon protease.

    PubMed

    Duman, Ramona E; Löwe, Jan

    2010-08-27

    Lon ATP-dependent proteases are key components of the protein quality control systems of bacterial cells and eukaryotic organelles. Eubacterial Lon proteases contain an N-terminal domain, an ATPase domain, and a protease domain, all in one polypeptide chain. The N-terminal domain is thought to be involved in substrate recognition, the ATPase domain in substrate unfolding and translocation into the protease chamber, and the protease domain in the hydrolysis of polypeptides into small peptide fragments. Like other AAA+ ATPases and self-compartmentalising proteases, Lon functions as an oligomeric complex, although the subunit stoichiometry is currently unclear. Here, we present crystal structures of truncated versions of Lon protease from Bacillus subtilis (BsLon), which reveal previously unknown architectural features of Lon complexes. Our analytical ultracentrifugation and electron microscopy show different oligomerisation of Lon proteases from two different bacterial species, Aquifex aeolicus and B. subtilis. The structure of BsLon-AP shows a hexameric complex consisting of a small part of the N-terminal domain, the ATPase, and protease domains. The structure shows the approximate arrangement of the three functional domains of Lon. It also reveals a resemblance between the architecture of Lon proteases and the bacterial proteasome-like protease HslUV. Our second structure, BsLon-N, represents the first 209 amino acids of the N-terminal domain of BsLon and consists of a globular domain, similar in structure to the E. coli Lon N-terminal domain, and an additional four-helix bundle, which is part of a predicted coiled-coil region. An unexpected dimeric interaction between BsLon-N monomers reveals the possibility that Lon complexes may be stabilised by coiled-coil interactions between neighbouring N-terminal domains. Together, BsLon-N and BsLon-AP are 36 amino acids short of offering a complete picture of a full-length Lon protease.

  2. Biased Signaling of Protease-Activated Receptors

    PubMed Central

    Zhao, Peishen; Metcalf, Matthew; Bunnett, Nigel W.

    2014-01-01

    In addition to their role in protein degradation and digestion, proteases can also function as hormone-like signaling molecules that regulate vital patho-physiological processes, including inflammation, hemostasis, pain, and repair mechanisms. Certain proteases can signal to cells by cleaving protease-activated receptors (PARs), a family of four G protein-coupled receptors. PARs are expressed by almost all cell types, control important physiological and disease-relevant processes, and are an emerging therapeutic target for major diseases. Most information about PAR activation and function derives from studies of a few proteases, for example thrombin in the case of PAR1, PAR3, and PAR4, and trypsin in the case of PAR2 and PAR4. These proteases cleave PARs at established sites with the extracellular N-terminal domains, and expose tethered ligands that stabilize conformations of the cleaved receptors that activate the canonical pathways of G protein- and/or β-arrestin-dependent signaling. However, a growing number of proteases have been identified that cleave PARs at divergent sites to activate distinct patterns of receptor signaling and trafficking. The capacity of these proteases to trigger distinct signaling pathways is referred to as biased signaling, and can lead to unique patho-physiological outcomes. Given that a different repertoire of proteases are activated in various patho-physiological conditions that may activate PARs by different mechanisms, signaling bias may account for the divergent actions of proteases and PARs. Moreover, therapies that target disease-relevant biased signaling pathways may be more effective and selective approaches for the treatment of protease- and PAR-driven diseases. Thus, rather than mediating the actions of a few proteases, PARs may integrate the biological actions of a wide spectrum of proteases in different patho-physiological conditions. PMID:24860547

  3. Proteolytic crosstalk in multi-protease networks

    NASA Astrophysics Data System (ADS)

    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.

  4. Fine Structure in 3C 120 and 3C 84. Ph.D. Thesis - Maryland Univ., 24 Aug. 1976

    NASA Technical Reports Server (NTRS)

    Hutton, L. K.

    1976-01-01

    Seven epochs of very long baseline radio interferometric observations of the Seyfert galaxies 3C 120 and 3C 84, at 3.8-cm wave length using stations at Westford, Massachusetts, Goldstone, California, Green Bank, West Virginia, and Onsala, Sweden, have been analyzed for source structure. An algorithm for reconstructing the brightness distribution of a spatially confined source from fringe amplitude and so called closure phase data has been developed and successfully applied to artificially generated test data and to data on the above mentioned sources. Over the two year time period of observation, 3C 120 was observed to consist of a double source showing apparent super relativistic expansion and separation velocities. The total flux changes comprising one outburst can be attributed to one of these components. 3C 84 showed much slower changes, evidently involving flux density changes in individual stationary components rather than relative motion.

  5. RXTE, VLBA, Optical, and Radio Monitoring of the Quasars 3C 279, PKS 1510--089, and 3C 273

    NASA Technical Reports Server (NTRS)

    Marscher, A. P.; Jorstad, S. G.; Aller, M. F.; McHardy, I. M.; Balonek, T. J.

    2001-01-01

    We are continuing our combined RXTE X-ray, VLBA imaging (at 43 GHz), optical (several observatories), and radio (University of Michigan Radio Astronomy Observatory) monitoring of the quasars 3C 279 and PKS 1510-089, and have started similar monitoring of 3C 273. X-ray flares in 3C 279 and PKS 1510-089 are associated with ejections of superluminal components. In addition, there is a close connection between the optical and X-ray variability of 3C 279. There is a strong correlation between the 14.5 GHz and X-ray variability of PKS 1510-089 in 1997 and 1998 (with the radio leading the X-ray) that becomes weaker in subsequent years. X-ray fluctuations occur on a variety of timescales in 3C 273, with a major prolonged outburst in mid-2001. The lead author will discuss the correlations in terms of inverse Compton models for the X-ray emission coupled with synchrotron models for the lower-frequency radiation. Synchrotron self-Compton models can explain the "reverse" time lag in PKS 1510-089 is well as the variable correlation between the X-ray variations and those at lower frequencies in this object and in 3C 279.

  6. Polarization-maintaining amplifier based on 3C fiber structures

    NASA Astrophysics Data System (ADS)

    Enokidani, Jun; Ito, Rumi; Sakurai, Tsutomu; Shin, Sumida; Tei, Kazuyoku

    2015-03-01

    Chirally-Coupled-Core (3C) fiber structure can preserve a single mode quality and even a linear polarization for a large core size. A principal advantage of fiber laser is its compatibility with monolithic integration and robust system. But so far, devices such as a combiner using the 3C fibers have not been reported. Here we report the first demonstration of such monolithic amplifier structure which contains an active fiber and a combiner based on 3C fibers. A single-stage amplifier is seeded by an EO Q-switched micro-laser and pumped by two high power fiber pigtailed 976-nm laser diodes via an in-house fabricated (2 + 1) × 1 pump signal combiner. The active fiber is based on a 3-m-long, 3C Yb-doped fiber (33 μm/250 μm core/cladding diameter with 0.06/0.46 NA). The amplifier demonstrates scaling up to 30W average power and 150 kW peak power in 0.3mJ, 2ns pulses. The beam profiles and beam qualities were characterized as its output power was varied up to 30W. The beam profile was maintained at a high beam quality of around M2=1.2. The spectral properties of the 3C fiber were also characterized as its output peak power was varied.

  7. Optical variability of PHL 1811 and 3C 273

    NASA Astrophysics Data System (ADS)

    Fan, J. H.; Kurtanidze, O.; Liu, Y.; Yuan, Y. H.; Hao, J. M.; Cai, W.; Xiao, H. B.; Pei, Z. Y.

    2015-03-01

    In this work, we reported the optical photometry monitoring results for two brightest nearby quasars, PHL 1811 and 3C 273 using the ST-6 camera at Abastumani Observatory, Georgia. For PHL 1811, we found 3 microvariability events with time scale of ΔT = 6.0 min. For 3C273, we found that the largest variations are ΔV = 0.369 +/- 0.028 mag, ΔR = 0.495 +/- 0.076 mag, and ΔI = 0.355 +/- 0.009 mag. When periodicity analysis methods are adopted to the available data, a period of p = 5.80 +/- 1.12 years is obtained for PHL 1811, and p = 21.10 +/- 0.14, 10.00 +/- 0.14, 7.30 +/- 0.09, 13.20 +/- 0.09, 2.10 +/- 0.06, and 0.68 +/- 0.05 years are obtained for 3C 273.

  8. Energetics and structural stability of Cs3C60

    SciTech Connect

    Saito, Susumu; Umemoto, Koichiro; Louie, Steven G.; Cohen, MarvinL.

    2003-12-15

    Using the ab initio pseudo potential total-energy method and the density-functional theory, we study the energetics of face-centered-cubic Cs3C60 which is a material of great interest as a possible high transition-temperature superconductor. At the optimized lattice constant the volume per C60 is found to be smaller than the most stable hexagon-coordination A15 phase, while the total energy of the fcc phase is about 0.9 eV higher than the A15 phase. These results indicate that a low-temperature and high-pressure synthesis method might be a possible way to produce the fcc Cs3C60 phase. In addition, it is also found that the A15 Cs3C60 should show a phase transformation from a hexagon-coordination phase to a pentagon-coordination phase under hydrostatic pressure.

  9. Graphene/3C-SiC Hybrid Nanolaminate.

    PubMed

    Zhuang, Hao; Yang, Bing; Heuser, Steffen; Huang, Nan; Fu, Haiyuan; Jiang, Xin

    2015-12-30

    In this work, we demonstrate a one-step approach to create graphene/3C-SiC nanolaminate structure using microwave plasma chemical vapor deposition technique. Layer-by-layer arrangement of thin 3C-SiC layers and graphene sheets is obtained with the thicknesses of the individual 3C-SiC layers and graphene sheets being 5-10 nm and 2-5 nm, respectively. An intimate contact between 3C-SiC and the graphene sheets is achieved and the nanolaminate film shows a high room temperature conductivity of 96.1 S/cm. A dedicated structural analysis of the nanolaminates by means of high-resolution transmission electron microscopy (HRTEM) reveals that the growth of the nanolaminates follows an iterative process: preferential graphene nucleation around the planar defects at the central region of the SiC layer, leading to the "splitting" of the SiC layer; and the thickening of the SiC layer after being "split". A growth mechanism based on both kinetics and thermodynamics is proposed. Following the proposed mechanism, it is possible to control the layer thickness of the graphene/3C-SiC hybrid nanolaminate by manipulating the carbon concentration in the gas phase, which is further experimentally verified. The high electrical conductivity, large surface area porous structure, feasible integration on different substrates (metal, Mo; semiconductor, Si and 2H-SiC; insulator, diamond) of the graphene/3C-SiC hybrid nanolaminate as well as other unprecedented advantages of the nanolaminate structure make it very promising for applications in mechanical, energy, and sensor-related areas. PMID:26650041

  10. Protease-degradable electrospun fibrous hydrogels

    NASA Astrophysics Data System (ADS)

    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.

  11. The linear polarization of 3C 345 in the ultraviolet

    NASA Technical Reports Server (NTRS)

    Dolan, Joseph F.; Boyd, Patricia T.; Wolinski, Karen G.; Smith, Paul S.; Impey, C. D.; Bless, Robert C.; Nelson, M. J.; Percival, J. W.; Taylor, M. J.; Elliot, J. L.

    1994-01-01

    The linear polarization of 3C 345, a superluminal radio source and OVV quasar, was observed in two bandpasses in the ultraviolet (centered at 2160 A and 2770 A) in 1993 April using the High Speed Photometer on the Hubble Space Telescope. The quasar is significantly polarized in the UV (p greater than 5%). Ground-based polarimetry was obtained 11 days later, but a difference in the position angle between the observations in the visible and those in the UV indicate that the magnitude of the polarization of 3C 345 may have changed over that time. If the two observation sets represent the same state of spectral polarization, then the large UV flux implies that either the polarization of the synchrotron continuum must stop decreasing in the UV, or that there is an additional source of polarized flux in the ultraviolet. Only if the UV observations represent a spectral polarization state with the same position angle in the visible seen previously in 3C 345 can the polarized flux be represented by a single power law consistent with the three-component model of Smith et al. This model consists of a polarized synchrotron component, an unpolarized component from the broad-line region, and an unpolarized component attributed to thermal radiation from an optically thick accretion disk. Additional simultaneous polarimetry in the UV and visible will be required to further constrain models of the continuum emission processes in 3C 345 and determine if the UV polarized flux is synchrotron in origin.

  12. Microwave Radiometer – 3 Channel (MWR3C) Handbook

    SciTech Connect

    Cadeddu, MP

    2012-05-04

    The microwave radiometer 3-channel (MWR3C) provides time-series measurements of brightness temperatures from three channels centered at 23.834, 30, and 89 GHz. These three channels are sensitive to the presence of liquid water and precipitable water vapor.

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

    NASA Astrophysics Data System (ADS)

    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.

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

    NASA Technical Reports Server (NTRS)

    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

  15. Mast cell proteases as pharmacological targets.

    PubMed

    Caughey, George H

    2016-05-01

    Mast cells are rich in proteases, which are the major proteins of intracellular granules and are released with histamine and heparin by activated cells. Most of these proteases are active in the granule as well as outside of the mast cell when secreted, and can cleave targets near degranulating mast cells and in adjoining tissue compartments. Some proteases released from mast cells reach the bloodstream and may have far-reaching actions. In terms of relative amounts, the major mast cell proteases include the tryptases, chymases, cathepsin G, carboxypeptidase A3, dipeptidylpeptidase I/cathepsin C, and cathepsins L and S. Some mast cells also produce granzyme B, plasminogen activators, and matrix metalloproteinases. Tryptases and chymases are almost entirely mast cell-specific, whereas other proteases, such as cathepsins G, C, and L are expressed by a variety of inflammatory cells. Carboxypeptidase A3 expression is a property shared by basophils and mast cells. Other proteases, such as mastins, are largely basophil-specific, although human basophils are protease-deficient compared with their murine counterparts. The major classes of mast cell proteases have been targeted for development of therapeutic inhibitors. Also, a human β-tryptase has been proposed as a potential drug itself, to inactivate of snake venins. Diseases linked to mast cell proteases include allergic diseases, such as asthma, eczema, and anaphylaxis, but also include non-allergic diseases such as inflammatory bowel disease, autoimmune arthritis, atherosclerosis, aortic aneurysms, hypertension, myocardial infarction, heart failure, pulmonary hypertension and scarring diseases of lungs and other organs. In some cases, studies performed in mouse models suggest protective or homeostatic roles for specific proteases (or groups of proteases) in infections by bacteria, worms and other parasites, and even in allergic inflammation. At the same time, a clearer picture has emerged of differences in the

  16. Gene expression and activity of digestive proteases in Daphnia: effects of cyanobacterial protease inhibitors

    PubMed Central

    2010-01-01

    Background The frequency of cyanobacterial blooms has increased worldwide, and these blooms have been claimed to be a major factor leading to the decline of the most important freshwater herbivores, i.e. representatives of the genus Daphnia. This suppression of Daphnia is partly attributed to the presence of biologically active secondary metabolites in cyanobacteria. Among these metabolites, protease inhibitors are found in almost every natural cyanobacterial bloom and have been shown to specifically inhibit Daphnia's digestive proteases in vitro, but to date no physiological responses of these serine proteases to cyanobacterial protease inhibitors in Daphnia have been reported in situ at the protein and genetic levels. Results Nine digestive proteases were detected in D. magna using activity-stained SDS-PAGE. Subsequent analyses by LC-MS/MS and database search led to the identification of respective protease genes. D. magna responded to dietary protease inhibitors by up-regulation of the expression of these respective proteases at the RNA-level and by the induction of new and less sensitive protease isoforms at the protein level. The up-regulation in response to dietary trypsin- and chymotrypsin-inhibitors ranged from 1.4-fold to 25.6-fold. These physiological responses of Daphnia, i.e. up-regulation of protease expression and the induction of isoforms, took place even after feeding on 20% cyanobacterial food for only 24 h. These physiological responses proved to be independent from microcystin effects. Conclusion Here for the first time it was shown in situ that a D. magna clone responds physiologically to dietary cyanobacterial protease inhibitors by phenotypic plasticity of the targets of these specific inhibitors, i.e. Daphnia gut proteases. These regulatory responses are adaptive for D. magna, as they increase the capacity for protein digestion in the presence of dietary protease inhibitors. The type and extent of these responses in protease expression might

  17. Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease.

    PubMed Central

    Kooi, C; Cox, A; Darling, P; Sokol, P A

    1994-01-01

    Pseudomonas cepacia produces at least two extracellular proteases with apparent molecular masses of 36,000 and 40,000 Da. The 36-kDa protease has high proteolytic activity and the 40-kDa protease has low proteolytic activity with hide powder azure as a substrate. Monoclonal antibodies (MAbs) were raised against the purified 36- and 40-kDa proteases. Several MAbs directed against the 36-kDa protease were found to recognize the 40-kDa protease by Western immunoblot analysis. Similarly, a MAb directed against the 40-kDa protease recognized the 36-kDa protease, suggesting that these two proteases may be immunologically related. A MAb directed against the 36-kDa protease, designated 36-6-8, and a MAb directed against the 40-kDa protease (MAb G-11) cross-reacted with other extracellular proteases, such as Pseudomonas aeruginosa elastase and alkaline protease, Pseudomonas pseudomallei protease, and the Vibrio cholerae hemagglutinin/protease. MAb 36-6-8 neutralized the P. cepacia 36-kDa protease, P. aeruginosa elastase, P. pseudomallei protease, and V. cholerae hemagglutinin/protease but did not affect P. aeruginosa alkaline protease activity. In contrast, MAb G-11 to the 40-kDa protease neutralized only the P. cepacia 36-kDa protease. This evidence suggests that the neutralizing MAb, 36-6-8, recognizes an epitope conserved among some metalloproteases. This epitope may lie at or near the active site of the P. cepacia 36-kDa protease and P. aeruginosa elastase. Images PMID:7516312

  18. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

  19. A biotechnology perspective of fungal proteases

    PubMed Central

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

    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 sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:26273247

  20. Extracellular proteases of Trichoderma species. A review.

    PubMed

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

    2005-01-01

    Cellulolytic, xylanolytic, chitinolytic and beta-1,3-glucanolytic enzyme systems of species belonging to the filamentous fungal genus Trichoderma have been investigated in details and are well characterised. The ability of Trichoderma strains to produce extracellular proteases has also been known for a long time, however, the proteolytic enzyme system is relatively unknown in this genus. Fortunately, in the recent years more and more attention is focused on the research in this field. The role of Trichoderma proteases in the biological control of plant pathogenic fungi and nematodes has been demonstrated, and it is also suspected that they may be important for the competitive saprophytic ability of green mould isolates and may represent potential virulence factors of Trichoderma strains as emerging fungal pathogens of clinical importance. The aim of this review is to summarize the information available about the extracellular proteases of Trichoderma. Numerous studies are available about the extracellular proteolytic enzyme profiles of Trichoderma strains and about the effect of abiotic environmental factors on protease activities. A number of protease enzymes have been purified to homogeneity and some protease encoding genes have been cloned and characterized. These results will be reviewed and the role of Trichoderma proteases in biological control as well as their advantages and disadvantages in biotechnology will be discussed. PMID:16003937

  1. A biotechnology perspective of fungal proteases.

    PubMed

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

    2015-06-01

    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 sauce production. Proteases can be produced in large quantities in a short time by established methods of fermentation. The parameters such as variation in C/N ratio, presence of some sugars, besides several other physical factors are important in the development of fermentation process. Proteases of fungal origin can be produced cost effectively, have an advantage faster production, the ease with which the enzymes can be modified and mycelium can be easily removed by filtration. The production of proteases has been carried out using submerged fermentation, but conditions in solid state fermentation lead to several potential advantages for the production of fungal enzymes. This review focuses on the production of fungal proteases, their distribution, structural-functional aspects, physical and chemical parameters, and the use of these enzymes in industrial applications. PMID:26273247

  2. Synthesis of C3/C1-Substituted Tetrahydroisoquinolines.

    PubMed

    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

  3. Cysteine Proteases from Bloodfeeding Arthropod Ectoparasites

    PubMed Central

    Sojka, Daniel; Francischetti, Ivo M. B.; Calvo, Eric; Kotsyfakis, Michalis

    2012-01-01

    Cysteine proteases have been discovered in various bloodfeeding ectoparasites. Here, we assemble the available information about the function of these peptidases and reveal their role in hematophagy and parasite development. While most of the data shed light on key proteolytic events that play a role in arthropod physiology, we also report on the association of cysteine proteases with arthropod vectorial capacity. With emphasis on ticks, specifically Ixodes ricinus, we finally propose a model about the contribution of cysteine peptidases to blood digestion, and how their concerted action with other tick midgut proteases leads to the absorbance of nutrients by the midgut epithelial cells. PMID:21660665

  4. HIV-1 Protease: Structure, Dynamics and Inhibition

    SciTech Connect

    Louis, John M.; Ishima, R.; Torchia, D.A.; Weber, Irene T.

    2008-06-03

    The HIV-1 protease is synthesized as part of a large Gag-Pol precursor protein. It is responsible for its own release from the precursor and the processing of the Gag and Gag-Pol polyproteins into the mature structural and functional proteins required for virus maturation. Because of its indispensable role, the mature HIV-1 protease dimer has proven to be a successful target for the development of antiviral agents. In the last 5 years, a major emphasis in protease research has been to improve inhibitor design and treatment regimens.

  5. X-Ray Monitoring of 3C120

    NASA Astrophysics Data System (ADS)

    Miller, Hugh

    The goal of this program is to characterize the variability at both X- ray and optical wavelengths simultaneously for the high luminosity, radio-loud AGN(RLAGN), 3C 120. The key question that is most central to the study of the variability of both RLAGN and radio-quiet AGN(RQAGN) is to identify a ''characteristic timescale", e.g. breaks (''knees") or spikes in the power density spectrum. There are now several RQAGN (e.g., NGC 3516 and Akn 120) for which characteristic timescales (i.e., knee frequencies) have been detected. However there is no RLAGN for which a knee frequency has been observed. The Seyfert-like RLAGN, 3C 120 offers a special opportunity for a possible first detection of a knee frequency in RLAGN.

  6. The double-lobed blazar 3C 371

    NASA Astrophysics Data System (ADS)

    Wrobel, J. M.; Lind, K. R.

    1990-01-01

    High dynamic range VLA imaging reveals, for the first time, that the blazar 3C 371 exhibits very low surface brightness twin radio lobes. These lobes straddle the dominant compact core and have a projected extent of about 42/h kpc. A weak component located in the western lobe shares the morphology, size, and inferred magnetic field configuration of radio source hot spots. A continuous wiggling jet connects this hot spot with the compact core. Double lobes, hot spots, and one-sided jets are traits of extended, edge-brightened radio sources. The discovery of corresponding components in 3C 371's extended emission suggests that this emission is an edge-brightened double, viewed at a large enough angle to the line of sight that the radio lobes do not overlap. A moderate viewing angle is consistent with 3C 371's low bolometric luminosity for a blazar. The diffuse radio halo that surrounds the twin lobes may be related to the fat bridges or bridge distortions shown by edge-brightened doubles with similar radio powers.

  7. Voltammetry of Sc{sub 3}@C{sub 82}

    SciTech Connect

    Anderson, M.R.; Dorn, H.C.; Stevenson, S.; Burbank, P.M.; Gibson, J.R.

    1997-01-15

    This paper describes the first electrochemical data for a polymetallic endohedral metallofullerene. The voltammetric behavior of Sc{sub 3}@C{sub 82} resembles that for La@C{sub 82} and Y@C{sub 82}, indicating that the identity of the metal does not influence dramatically the energies of the metallofullerene molecular orbitals. The three Sc atoms, however, transfer more electrons from the encapsulated metals to the cage than the monometallic counterparts, accounting for the differences in the voltammetry for Sc{sub 3}@C{sub 82}. The similarity of the voltammetry of Sc{sub 3}@C{sub 82} with that of the monometallic fullerenes suggests that Sc{sub 3}{sup +4}@C{sub 82}{sup 4-} may describe the formal charges on the Sc trimer and the C{sub 82} cage of these species. Metallofullerene electronic structure and physical properties should be better understood as more metallofullerenes become available for investigation. 31 refs., 1 fig., 1 tab.

  8. AGN feedback on the ISM of 3C 236

    NASA Astrophysics Data System (ADS)

    Labiano, A.; García-Burillo, S.; Combes, F.; Usero, A.; Soria-Ruiz, R.; Tremblay, G.; Neri, R.; Fuente, A.; Morganti, R.; Oosterloo, T.

    2013-03-01

    We have carried out 1mm/3mm continuum and 12CO(2-1) line high resolution observations to identify the footprints of AGN feedback on 3C 236. The CO emission comes from a spatially resolved disk characterized by a regular rotating pattern. Within the limits imposed by the sensitivity and velocity coverage of our data, we do not detect any outflow signatures in the cold molecular gas. Re-inspection of optical and IR spectra, shows the presence of a previously unknown ionized gas outflow. The star-formation efficiency in 3C 236, is consistent with the value measured in normal galaxies, which follow the canonical Kennicutt-Schmidt law. This result, confirmed to hold in other young radio sources examined in this work, is in stark contrast with the factor of 10-50 lower SFE that has been claimed to characterize evolved powerful radio galaxies. The recent reactivation of the AGN in 3C 236 is a likely explanation for the early evolutionary status of its molecular disk.

  9. The milliarcsecond structure of 3C 273 at 22 GHz

    SciTech Connect

    Zensus, J.A.; Biretta, J.A.; Unwin, S.C.; Cohen, M.H. Owens Valley Radio Observatory, Pasadena, CA )

    1990-12-01

    The first VLBI images at 22 GHz of the jet in the quasar 3C 273 are presented. In addition to the compact core region, two emission regions can be identified with features seen at lower frequencies; they separate from the core with constant speeds of 0.65 + or - 0.09 and 0.92 + or - 0.11 mas/yr, corresponding to apparent superluminal motion of 4.3 + or - 0.3c and 6.1 + or - 0.3c (for Ho = 100 km/s Mpc, qo = 0.5). The core region brightened at about the estimated epoch of zero separation for the latest superluminal component, suggesting a causal relationship. The curved ridge line of the jet smoothly extends inward towards the core, although no pronounced bends in the range of core distance 0.5-2.5 mas are seen. No significant evidence is found against a common path of subsequent superluminal features. An apparent frequency dependence in the position of one superluminal feature tentatively suggests that opacity effects across the jet direction are present. The results are consistent with an interpretation of the superluminal features as shocks in an underlying relativistic flow, although alternative explanations cannot be ruled out. 43 refs.

  10. The Trails of Superluminal Jet Components in 3C 111

    NASA Technical Reports Server (NTRS)

    Kadler, M.; Ros, E.; 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.

    2007-01-01

    The parsec-scale radio jet of the broad-line radio galaxy 3C 111 has been monitored since 1995 as part of the 2cm Survey and MOJAVE monitoring observations conducted with the VLBA. Here, we present results from 18 epochs of VLBA observations of 3C 111 and from 18 years of radio flux density monitoring observations conducted at the University of Michigan. A major radio flux-density outburst of 3C 111 occurred in 1996 and was followed by a particularly bright plasma ejection associated with a superluminal jet component. This major event allows us to study a variety of processes associated with outbursts of radio-loud AGN in much greater detail than possible in other cases: the primary perturbation gives rise to the formation of a forward and a backward-shock, which both evolve in characteristically different ways and allow us to draw conclusions about the workflow of jet-production events; the expansion, acceleration and recollimation of the ejected jet plasma in an environment with steep pressure and density gradients are revealed; trailing components are formed in the wake of the primary perturbation as a result of Kelvin- Helmholtz instabilities from the interaction of the jet with the external medium. The jet-medium interaction is further scrutinized by the linear-polarization signature of jet components traveling along the jet and passing a region of steep pressure/density gradients.

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

    SciTech Connect

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

    1982-08-15

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

  12. Human immunodeficiency virus 1 protease expressed in Escherichia coli behaves as a dimeric aspartic protease.

    PubMed Central

    Meek, T D; Dayton, B D; Metcalf, B W; Dreyer, G B; Strickler, J E; Gorniak, J G; Rosenberg, M; Moore, M L; Magaard, V W; Debouck, C

    1989-01-01

    Recombinant human immunodeficiency virus 1 (HIV-1) protease, purified from a bacterial expression system, processed a recombinant form of its natural substrate, Pr55gag, into protein fragments that possess molecular weights commensurate with those of the virion gag proteins. Molecular weights of the protease obtained under denaturing and nondenaturing conditions (11,000 and 22,000, respectively) and chemical crosslinking studies were consistent with a dimeric structure for the active enzyme. The protease appropriately cleaved the nonapeptide Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 between the tyrosine and proline residues. HIV-1 protease was sensitive to inactivators of the aspartic proteases. The aspartic protease inactivator 1,2-epoxy-3-(4-nitrophenoxy)propane produced irreversible, time-dependent inactivation of the protease. The pH-dependent kinetics of this inactivator were consistent with the requirement of an unprotonated carboxyl group in the active site of the enzyme, suggesting that HIV-1 protease is also an aspartic protease. Images PMID:2648384

  13. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application. PMID:27137097

  14. Secreted fungal aspartic proteases: A review.

    PubMed

    Mandujano-González, Virginia; Villa-Tanaca, Lourdes; Anducho-Reyes, Miguel Angel; Mercado-Flores, Yuridia

    2016-01-01

    The aspartic proteases, also called aspartyl and aspartate proteases or acid proteases (E.C.3.4.23), belong to the endopeptidase family and are characterized by the conserved sequence Asp-Gly-Thr at the active site. These enzymes are found in a wide variety of microorganisms in which they perform important functions related to nutrition and pathogenesis. In addition, their high activity and stability at acid pH make them attractive for industrial application in the food industry; specifically, they are used as milk-coagulating agents in cheese production or serve to improve the taste of some foods. This review presents an analysis of the characteristics and properties of secreted microbial aspartic proteases and their potential for commercial application.

  15. Vanadium inhibition of serine and cysteine proteases.

    PubMed

    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.

  16. Bioinformatics of proteases in the MEROPS database.

    PubMed

    Barrett, Alan J

    2004-05-01

    Proteolytic enzymes represent approximately approximately 2% of the total number of proteins present in all types of organisms. Many of these enzymes are of medical importance, and those that are of potential interest as drug targets can be divided into the endogenous enzymes encoded in the human genome, and the exogenous proteases encoded in the genomes of disease-causing organisms. There are also naturally occurring inhibitors of proteases, some of which have pharmaceutical relevance. The MEROPS database provides a rich source of information on proteases and their inhibitors. Storage and retrieval of this information is facilitated by the use of a hierarchical classification system (which was pioneered by the compilers of the database) in which homologous proteases and their inhibitors are divided into clans and families. PMID:15216937

  17. Electrically sensing protease activity with nanopores

    NASA Astrophysics Data System (ADS)

    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.

  18. Assimilatory Sulfate Reduction in C3, C3-C4, and C4 Species of Flaveria1

    PubMed Central

    Koprivova, Anna; Melzer, Michael; von Ballmoos, Peter; Mandel, Therese; Brunold, Christian; Kopriva, Stanislav

    2001-01-01

    The activity of the enzymes catalyzing the first two steps of sulfate assimilation, ATP sulfurylase and adenosine 5′-phosphosulfate reductase (APR), are confined to bundle sheath cells in several C4 monocot species. With the aim to analyze the molecular basis of this distribution and to determine whether it was a prerequisite or a consequence of the C4 photosynthetic mechanism, we compared the intercellular distribution of the activity and the mRNA of APR in C3, C3-C4, C4-like, and C4 species of the dicot genus Flaveria. Measurements of APR activity, mRNA level, and protein accumulation in six Flaveria species revealed that APR activity, cysteine, and glutathione levels were significantly higher in C4-like and C4 species than in C3 and C3-C4 species. ATP sulfurylase and APR mRNA were present at comparable levels in both mesophyll and bundle sheath cells of C4 species Flaveria trinervia. Immunogold electron microscopy demonstrated the presence of APR protein in chloroplasts of both cell types. These findings, taken together with results from the literature, show that the localization of assimilatory sulfate reduction in the bundle sheath cells is not ubiquitous among C4 plants and therefore is neither a prerequisite nor a consequence of C4 photosynthesis. PMID:11598228

  19. HIV Protease Inhibitors and Obesity

    PubMed Central

    Anuurad, Erdembileg; Bremer, Andrew; Berglund, Lars

    2011-01-01

    Purpose of review To review the current scientific literature and recent clinical trials on HIV protease inhibitors (PIs) and their potential role in the pathogenesis of lipodystrophy and metabolic disorders. Recent findings HIV PI treatment may affect the normal stimulatory effect of insulin on glucose and fat storage. Further, chronic inflammation from HIV infection and PI treatment trigger cellular homeostatic stress responses with adverse effects on intermediary metabolism. The physiologic outcome is such that total adipocyte storage capacity is decreased, and the remaining adipocytes resist further fat storage. This process leads to a pathologic cycle of lipodystrophy and lipotoxicity, a pro-atherogenic lipid profile, and a clinical phenotype of increased central body fat distribution similar to the metabolic syndrome. Summary PIs are a key component of antiretroviral therapy and have dramatically improved the life expectancy of HIV-infected individuals. However, they are also associated with abnormalities in glucose/lipid metabolism and body fat distribution. Further studies are needed to better define the pathogenesis of PI-associated metabolic and body fat changes and their potential treatment. PMID:20717021

  20. ADAM Proteases and Gastrointestinal Function.

    PubMed

    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.

  1. Neutral serine proteases of neutrophils.

    PubMed

    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

  2. ADAM Proteases and Gastrointestinal Function

    PubMed Central

    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

  3. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    SciTech Connect

    Wartchow, C.A.; Wang, Peng; Bednarski, M.D.; Callstrom, M.R. |

    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.

  4. Inhibition of Norovirus 3CL Protease by Bisulfite Adducts of Transition State Inhibitors

    PubMed Central

    Mandadapu, Sivakoteswara Rao; Gunnam, Mallikarjuna Reddy; Tiew, Kok-Chuan; Uy, Roxanne Adeline Z.; Prior, Allan M.; Alliston, Kevin R.; Hua, Duy H.; Kim, Yunjeong; Chang, Kyeong-Ok; Groutas, William C.

    2013-01-01

    Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the U.S. alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED50 of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters. PMID:23218713

  5. Inhibition of norovirus 3CL protease by bisulfite adducts of transition state inhibitors.

    PubMed

    Mandadapu, Sivakoteswara Rao; Gunnam, Mallikarjuna Reddy; Tiew, Kok-Chuan; Uy, Roxanne Adeline Z; Prior, Allan M; Alliston, Kevin R; Hua, Duy H; Kim, Yunjeong; Chang, Kyeong-Ok; Groutas, William C

    2013-01-01

    Noroviruses are the most common cause of acute viral gastroenteritis, accounting for >21 million cases annually in the US alone. Norovirus infections constitute an important health problem for which there are no specific antiviral therapeutics or vaccines. In this study, a series of bisulfite adducts derived from representative transition state inhibitors (dipeptidyl aldehydes and α-ketoamides) was synthesized and shown to exhibit anti-norovirus activity in a cell-based replicon system. The ED(50) of the most effective inhibitor was 60 nM. This study demonstrates for the first time the utilization of bisulfite adducts of transition state inhibitors in the inhibition of norovirus 3C-like protease in vitro and in a cell-based replicon system. The approach described herein can be extended to the synthesis of the bisulfite adducts of other classes of transition state inhibitors of serine and cysteine proteases, such as α-ketoheterocycles and α-ketoesters.

  6. The optical variability of the quasar 3C 446

    SciTech Connect

    Barbieri, C.; Vio, R.; Cappellaro, E; Turatto, M Padova Osservatorio Astronomico, Padua )

    1990-08-01

    The optical variability of the quasar 3C 446 is investigated using power spectrum and structure function analysis along with a new set of observations that extend the available data till 1989. No contradiction is found between the PS and SF analyses. The presence of the 1540-day periodicity is strengthened by the occurrence of the 1988 luminosity peak, suggesting that the next burst will occur in the northern spring of 1992. The time series of the quasar is nonstationary. The light variations are determined by a sequence of luminosity bursts, mostly regularly spaced in time and lasting up to 2 yr. 25 refs.

  7. SUZAKU OBSERVATION OF THE GIANT RADIO GALAXY 3C 326

    SciTech Connect

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

    2009-11-20

    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 and 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 modified with the Galactic absorption. The photon index and 1 keV flux density were derived as GAMMA = 1.82{sup +0.26}{sub -0.24} +- 0.04 and S {sub X} = 19.4{sup +3.3}{sub -3.2} +- 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 (IC) 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 IC X-ray emission. A comparison of the radio to X-ray fluxes yields the energy densities of electron and magnetic field as u{sub e} = (2.3 +- 0.3 +- 0.3) x 10{sup -13} erg cm{sup -3} and u{sub m} = (1.2{sup +0.2}{sub -0.1} +- 0.2) x 10{sup -14} erg cm{sup -3}, respectively. The galaxy is suggested to host a low-luminosity nucleus with an absorption-corrected 2-10 keV luminosity of <2 x 10{sup 42} erg s{sup -1}, together with a relatively weak radio core. The energetics in the west lobe of 3C 326 were compared with those of moderate radio galaxies with a size of approx100 kpc. The west lobe of 3C 326 is confirmed to agree with the correlations for the moderate radio galaxies, u{sub e} propor to D {sup -2.2+}-{sup 0.4} and u{sub m} propor to D {sup -2.4+}-{sup 0.4}, where D is their total physical size. This implies that the lobes of 3C 326 are still being energized by the jet, despite the current weakness of the nuclear activity.

  8. Protease Inhibitors Targeting Coronavirus and Filovirus Entry

    PubMed Central

    Zhou, Yanchen; Vedantham, Punitha; Lu, Kai; Agudelo, Juliet; Carrion, Ricardo; Nunneley, Jerritt W.; Barnard, Dale; Pöhlmann, Stefan; McKerrow, James H.; Renslo, Adam R.; Simmons, Graham

    2016-01-01

    In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess, whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and

  9. New soluble ATP-dependent protease, Ti, in Escherichia coli that is distinct from protease La

    SciTech Connect

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

    1987-05-01

    E. coli must contain other ATP-requiring proteolytic systems in addition to protease La (the lon gene product). A new ATP-dependent protease was purified from lon cells which lack protease La, as shown by immuno-blotting. This enzyme hydrolyzes (TH)casein to acid-soluble products in the presence of ATP (or dATP) and MgS . Nonhydrolyzable ATP analogs, other nucleoside triphosphates and AMP can not replace ATP. Therefore, ATP hydrolysis appears necessary for proteolysis. The enzyme appears to be a serine protease, but also contains essential thiol residues. Unlike protease La, it is not inhibited by vanadate, heparin, or the defective R9 subunit of protease La. On gel filtration, this enzyme has an apparent Mr of 340,000 and is comprised of two components of 190,000D and 130,000D, which can be separated by phosphocellulose chromatography. By themselves, these components do not show ATP-dependent proteolysis, but when mixed, full activity is restored. These finding and similar ones of Maurizi and Gottesman indicate that E. coli contain two soluble ATP-dependent proteases, which function by different mechanisms. This new enzyme may contribute to the rapid breakdown of abnormal polypeptides or of normal proteins during starvation. The authors propose to name it protease Ti.

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

    SciTech Connect

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

    1987-08-01

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

  11. Multiwavelength variability analysis of the FSRQ 3C 279

    NASA Astrophysics Data System (ADS)

    Patiño-Álvarez, V.; Chavushyan, V.; León-Tavares, J.; Carramiñana, A.; Carrasco, L.; Fernandes, S.; Schlegel, E. M.; López-Rodríguez, E.

    2015-03-01

    We present a multifrequency analysis of the variability in the flat-spectrum radio quasar 3C 279 from 2008 to 2014. Our multiwavelength dataset includes gamma-ray data from Fermi/LAT (Abdo et al. 2009), observations in 1mm from SMA (Gurwell et al. 2007), Near Infrared from OAGH (Carramiñana & Carrasco 2009) and SMARTS (Bonning et al. 2012); optical V band from the Steward Observatory (Smith et al. 2009) and SMARTS; optical spectra from OAGH (Patiño-Álvarez et al. 2013) and the Steward Observatory; and polarization spectra from the Steward Observatory. The light curves are shown in Fig. 1. Six out of seven optical activity periods identified within our dataset show clear counterparts in mm, NIR and gamma-rays, however, the late 2011 - early 2012 optical flare does not have a counterpart in the GeV regime. In this contribution, we discuss the flaring evolution of 3C 279 and speculate about the production of the anomalous activity period.

  12. Shocked Molecular Gas in the SNR 3C391

    NASA Astrophysics Data System (ADS)

    Bock, D. C.-J.; Wright, M. C. H.; Frail, D. A.; Gaensler, B. M.; Wilner, D. J.

    1999-05-01

    The discovery of OH (1720 MHz) masers in supernova remnants (SNRs) provides us with a convenient method for identifying promising candidates for studies of SNR/molecular cloud interactions. One remnant which is an ideal subject is 3C391. Here, single dish observations reveal a myriad of molecular lines from a giant shocked cloud at the blast-wave of the remnant. We present recent millimeter-wave observations of 3C391 with the Berkeley-Illinois-Maryland Association (BIMA) array. We have imaged the region towards the OH maser in HCN (J=1--0) and HCO+ (J=1--0) at a resolution of 4 arcsec. The morphology and brightness of the emission in HCO+ and HCN are similar; we resolve the shocked cloud seen by Frail and Mitchell (1998) into several clumps of characteristic size 10 arcsec. The maser is midway between two of these clumps, rather than being coincident with the peak column density. The clumps all have similar spectra with line-widths of ~ 30 km s(-1) , although there is evidence for a velocity gradient between the clumps. We examine the physical conditions of the shocked environment and discuss the relative abundances of the molecules. A companion study of the interaction between the SNR W51C and an associated molecular cloud is underway.

  13. The enterovirus protease inhibitor rupintrivir exerts cross-genotypic anti-norovirus activity and clears cells from the norovirus replicon.

    PubMed

    Rocha-Pereira, J; Nascimento, M S J; Ma, Q; Hilgenfeld, R; Neyts, J; Jochmans, D

    2014-08-01

    Potent and safe inhibitors of norovirus replication are needed for the treatment and prophylaxis of norovirus infections. We here report that the in vitro anti-norovirus activity of the protease inhibitor rupintrivir is extended to murine noroviruses and that rupintrivir clears human cells from their Norwalk replicon after only two passages of antiviral pressure. In addition, we demonstrate that rupintrivir inhibits the human norovirus (genogroup II [GII]) protease and further explain the inhibitory effect of the molecule by means of molecular modeling on the basis of the crystal structure of the Norwalk virus protease. The combination of rupintrivir with the RNA-dependent RNA polymerase inhibitors 2'-C-methylcytidine and favipiravir (T-705) resulted in a merely additive antiviral effect. The fact that rupintrivir is active against noroviruses belonging to genogroup I (Norwalk virus), genogroup V (murine norovirus), and the recombinant 3C-like protease of a GII norovirus suggests that the drug exerts cross-genotypic anti-norovirus activity and will thus most likely be effective against the clinically relevant human norovirus strains. The design of antiviral molecules targeting the norovirus protease could be a valuable approach for the treatment and/or prophylaxis of norovirus infections.

  14. 17 CFR 270.3c-4 - Definition of “common trust fund” as used in section 3(c)(3) of the Act.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 17 Commodity and Securities Exchanges 3 2010-04-01 2010-04-01 false Definition of âcommon trust... 1940 § 270.3c-4 Definition of “common trust fund” as used in section 3(c)(3) of the Act. The term common trust fund as used in section 3(c)(3) of the Act (15 U.S.C. 80a-3(c)(3)) shall include a...

  15. Production of alkaline protease from Cellulosimicrobium cellulans

    PubMed Central

    Ferracini-Santos, Luciana; Sato, Hélia H

    2009-01-01

    Cellulosimicrobium cellulans is one of the microorganisms that produces a wide variety of yeast cell wall-degrading enzymes, β-1,3-glucanase, protease and chitinase. Dried cells of Saccharomyces cerevisiae were used as carbon and nitrogen source for cell growth and protease production. The medium components KH2PO4, KOH and dried yeast cells showed a significant effect (p<0.05) on the factorial fractional design. A second design was prepared using two factors: pH and percentage of dried yeast cells. The results showed that the culture medium for the maximum production of protease was 0.2 g/l of MgSO4.7H2O, 2.0 g/l of (NH4)2SO4 and 8% of dried yeast cells in 0.15M phosphate buffer at pH 8.0. The maximum alkaline protease production was 7.0 ± 0.27 U/ml over the center point. Crude protease showed best activity at 50ºC and pH 7.0-8.0, and was stable at 50ºC. PMID:24031317

  16. Protease Inhibitors from Plants with Antimicrobial Activity

    PubMed Central

    Kim, Jin-Young; Park, Seong-Cheol; Hwang, Indeok; Cheong, Hyeonsook; Nah, Jae-Woon; Hahm, Kyung-Soo; Park, Yoonkyung

    2009-01-01

    Antimicrobial proteins (peptides) are known to play important roles in the innate host defense mechanisms of most living organisms, including plants, insects, amphibians and mammals. They are also known to possess potent antibiotic activity against bacteria, fungi, and even certain viruses. Recently, the rapid emergence of microbial pathogens that are resistant to currently available antibiotics has triggered considerable interest in the isolation and investigation of the mode of action of antimicrobial proteins (peptides). Plants produce a variety of proteins (peptides) that are involved in the defense against pathogens and invading organisms, including ribosome-inactivating proteins, lectins, protease inhibitors and antifungal peptides (proteins). Specially, the protease inhibitors can inhibit aspartic, serine and cysteine proteinases. Increased levels of trypsin and chymotrypsin inhibitors correlated with the plants resistance to the pathogen. Usually, the purification of antimicrobial proteins (peptides) with protease inhibitor activity was accomplished by salt-extraction, ultrafiltration and C18 reverse phase chromatography, successfully. We discuss the relation between antimicrobial and anti-protease activity in this review. Protease inhibitors from plants potently inhibited the growth of a variety of pathogenic bacterial and fungal strains and are therefore excellent candidates for use as the lead compounds for the development of novel antimicrobial agents. PMID:19582234

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

    Code of Federal Regulations, 2010 CFR

    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...

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

    NASA Astrophysics Data System (ADS)

    Roberts, David H.; Kollgaard, Ronald I.; Brown, Leslie F.; Gabuzda, Denise C.; Wardle, John F. C.

    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.

  19. Trisodium citrate, Na3(C6H5O7)

    PubMed Central

    Rammohan, Alagappa; Kaduk, James A.

    2016-01-01

    The crystal structure of anhydrous tris­odium citrate, Na3(C6H5O7), has been solved and refined using synchrotron X-ray powder diffraction data, and optimized using density functional theory (DFT). There are two independent five-coordinate Na+ and one six-coordinate Na+ cations in the asymmetric unit. The [NaO5] and [NaO6] polyhedra share edges and corners to form a three-dimensional framework. There are channels parallel to the a and b axes in which the remainder of the citrate anions reside. The only hydrogen bonds are an intra­molecular one between the hy­droxy group and one of the terminal carboxyl­ate O atoms and an intermolecular one between a methylene group and the hydroxyl O atom. PMID:27308044

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

    NASA Astrophysics Data System (ADS)

    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 <γ< -2.6. Conclusions: 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.

  1. Serine protease inhibitors of parasitic helminths.

    PubMed

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

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships. PMID:22310379

  2. Coagulation, Protease Activated Receptors and Viral Myocarditis

    PubMed Central

    Antoniak, Silvio; Mackman, Nigel

    2013-01-01

    The coagulation protease cascade plays an essential role in hemostasis. In addition, a clot contributes to host defense by limiting the spread of pathogens. Coagulation proteases induce intracellular signaling by cleavage of cell surface receptors called protease-activated receptors (PARs). These receptors allow cells to sense changes in the extracellular environment, such as infection. Viruses activate the coagulation cascade by inducing tissue factor expression and by disrupting the endothelium. Virus infection of the heart can cause myocarditis, cardiac remodeling and heart failure. Recent studies using a mouse model have shown that tissue factor, thrombin and PAR-1 signaling all positively regulate the innate immune during viral myocarditis. In contrast, PAR-2 signaling was found to inhibit interferon-β expression and the innate immune response. These observations suggest that anticoagulants may impair the innate immune response to viral infection and that inhibition of PAR-2 may be a new target to reduce viral myocarditis.. PMID:24203054

  3. Subtilisin-like proteases in nematodes.

    PubMed

    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

  4. Conformational selection in trypsin-like proteases

    PubMed Central

    Pozzi, Nicola; Vogt, Austin D.; Gohara, David W.; Di Cera, Enrico

    2012-01-01

    For over four decades, two competing mechanisms of ligand recognition – conformational selection and induced-fit - have dominated our interpretation of protein allostery. Defining the mechanism broadens our understanding of the system and impacts our ability to design effective drugs and new therapeutics. Recent kinetics studies demonstrate that trypsin-like proteases exist in equilibrium between two forms: one fully accessible to substrate (E) and the other with the active site occluded (E*). Analysis of the structural database confirms existence of the E* and E forms and vouches for the allosteric nature of the trypsin fold. Allostery in terms of conformational selection establishes an important paradigm in the protease field and enables protein engineers to expand the repertoire of proteases as therapeutics. PMID:22664096

  5. Dataset of cocoa aspartic protease cleavage sites.

    PubMed

    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

  6. Nidovirus papain-like proteases: multifunctional enzymes with protease, deubiquitinating and deISGylating activities

    PubMed Central

    Mielech, Anna M.; Chen, Yafang; Mesecar, Andrew D.; Baker, Susan C.

    2014-01-01

    Coronaviruses and arteriviruses, members of the order Nidovirales, are positive strand RNA viruses that encode large replicase polyproteins that are processed by viral proteases to generate the nonstructural proteins which mediate viral RNA synthesis. The viral papain-like proteases (PLPs) are critical for processing the amino-terminal end of the replicase and are attractive targets for antiviral therapies. With the analysis of the papain-like protease of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), came the realization of the multifunctional nature of these enzymes. Structural and enzymatic studies revealed that SARS-CoV PLpro can act as both a protease to cleave peptide bonds and also as a deubiquitinating (DUB) enzyme to cleave the isopeptide bonds found in polyubiquitin chains. Furthermore, viral DUBs can also remove the protective effect of conjugated ubiquitin-like molecules such as interferon stimulated gene 15 (ISG15). Extension of these studies to other coronaviruses and arteriviruses led to the realization that viral protease/DUB activity is conserved in many family members. Overexpression studies revealed that viral protease/DUB activity can modulate or block activation of the innate immune response pathway. Importantly, mutations that alter DUB activity but not viral protease activity have been identified and arteriviruses expressing DUB mutants stimulated higher levels of acute inflammatory cytokines after infection. Further understanding of the multifunctional nature of the Nidovirus PLP/DUBs may facilitate vaccine development. Here, we review studies describing the PLPs’ enzymatic activity and their role in virus pathogenesis. PMID:24512893

  7. 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

    PubMed Central

    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

  8. 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

    DOE PAGES

    Galasiti Kankanamalage, Anushka C.; 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; et al

    2015-04-09

    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 antinorovirus 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.

  9. Serine Protease(s) Secreted by the Nematode Trichuris muris Degrade the Mucus Barrier

    PubMed Central

    Hasnain, Sumaira Z.; McGuckin, Michael A.; Grencis, Richard K.; Thornton, David J.

    2012-01-01

    The polymeric mucin component of the intestinal mucus barrier changes during nematode infection to provide not only physical protection but also to directly affect pathogenic nematodes and aid expulsion. Despite this, the direct interaction of the nematodes with the mucins and the mucus barrier has not previously been addressed. We used the well-established Trichuris muris nematode model to investigate the effect on mucins of the complex mixture of immunogenic proteins secreted by the nematode called excretory/secretory products (ESPs). Different regimes of T. muris infection were used to simulate chronic (low dose) or acute (high dose) infection. Mucus/mucins isolated from mice and from the human intestinal cell line, LS174T, were treated with ESPs. We demonstrate that serine protease(s) secreted by the nematode have the ability to change the properties of the mucus barrier, making it more porous by degrading the mucin component of the mucus gel. Specifically, the serine protease(s) acted on the N-terminal polymerising domain of the major intestinal mucin Muc2, resulting in depolymerisation of Muc2 polymers. Importantly, the respiratory/gastric mucin Muc5ac, which is induced in the intestine and is critical for worm expulsion, was protected from the depolymerising effect exerted by ESPs. Furthermore, serine protease inhibitors (Serpins) which may protect the mucins, in particular Muc2, from depolymerisation, were highly expressed in mice resistant to chronic infection. Thus, we demonstrate that nematodes secrete serine protease(s) to degrade mucins within the mucus barrier, which may modify the niche of the parasite to prevent clearance from the host or facilitate efficient mating and egg laying from the posterior end of the parasite that is in intimate contact with the mucus barrier. However, during a TH2-mediated worm expulsion response, serpins, Muc5ac and increased levels of Muc2 protect the barrier from degradation by the nematode secreted protease(s). PMID

  10. Current and Novel Inhibitors of HIV Protease

    PubMed Central

    Pokorná, Jana; Machala, Ladislav; Řezáčová, Pavlína; Konvalinka, Jan

    2009-01-01

    The design, development and clinical success of HIV protease inhibitors represent one of the most remarkable achievements of molecular medicine. This review describes all nine currently available FDA-approved protease inhibitors, discusses their pharmacokinetic properties, off-target activities, side-effects, and resistance profiles. The compounds in the various stages of clinical development are also introduced, as well as alternative approaches, aiming at other functional domains of HIV PR. The potential of these novel compounds to open new way to the rational drug design of human viruses is critically assessed. PMID:21994591

  11. Using specificity to strategically target proteases

    PubMed Central

    Lim, Mark D.; Craik, Charles S.

    2009-01-01

    Proteases are a family of naturally occurring enzymes in the body whose dysregulation has been implicated in numerous diseases and cancers. Their ability to selectively and catalytically turnover substrate adds both signal amplification and functionality as parameters for the detection of disease. This review will focus on the development of activity-based methodologies to characterize proteases, and in particular, the use of positional scanning, synthetic combinatorial libraries (PS-SCL’s), and substrate activity screening (SAS) assays. The use of these approaches to better understand a protease’s natural substrate will be discussed as well as the technologies that emerged. PMID:18434168

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

    DOEpatents

    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.

  13. The Aligned z~1 Radio Galaxy 3C 280

    NASA Astrophysics Data System (ADS)

    Ridgway, Susan E.; Stockton, Alan; Lacy, Mark

    2004-01-01

    The z~1 radio galaxy 3C 280 has a particularly striking rest-frame UV morphology, with multiple line and continuum components precisely aligned with the radio structure, including an obvious semicircular arc. Here we explore the nature of these various components by bringing together Hubble Space Telescope and ground-based imaging, ground-based spectroscopy, and radio mapping. From plausible decompositions of the spectra, we show that the continuum of the nuclear component is likely dominated by a combination of nebular thermal continuum, quasar light, and light from old stars. A component that falls directly on the probable path of the radio jet shows mostly nebular thermal continuum and includes contributions from a relatively young stellar population with age around 100 Myr. The arc appears to be completely dominated by line emission and nebular thermal continuum, with no evidence for a significant stellar contribution. Although much of the aligned light is in UV components, the underlying old elliptical galaxy is also well aligned with the radio axis. The elliptical galaxy is well fitted by a de Vaucouleurs profile, probably has a moderately old stellar population (~3 Gyr), and is a massive system with a velocity dispersion of σ~270 km s-1 that implies that it contains a supermassive black hole. Although the arc and the extended emission surrounding the eastern lobe suggest that interactions between the radio lobe and jet must have been important in creating the UV morphology, the ionization and kinematic properties in these components are more consistent with photoionization than shock excitation. 3C 280 may be a transition object between the compact steep-spectrum radio galaxies, which seem to be shock-dominated, and the extended radio sources, which may have evolved past this phase and rarely show shock signatures. Based on observations made with the NASA/ESA Hubble Space Telescope, obtained at the Space Telescope Science Institute, which is operated by the

  14. Transient ECM protease activity promotes synaptic plasticity

    PubMed Central

    Magnowska, Marta; Gorkiewicz, Tomasz; Suska, Anna; Wawrzyniak, Marcin; Rutkowska-Wlodarczyk, Izabela; Kaczmarek, Leszek; Wlodarczyk, Jakub

    2016-01-01

    Activity-dependent proteolysis at a synapse has been recognized as a pivotal factor in controlling dynamic changes in dendritic spine shape and function; however, excessive proteolytic activity is detrimental to the cells. The exact mechanism of control of these seemingly contradictory outcomes of protease activity remains unknown. Here, we reveal that dendritic spine maturation is strictly controlled by the proteolytic activity, and its inhibition by the endogenous inhibitor (Tissue inhibitor of matrix metalloproteinases-1 – TIMP-1). Excessive proteolytic activity impairs long-term potentiation of the synaptic efficacy (LTP), and this impairment could be rescued by inhibition of protease activity. Moreover LTP is altered persistently when the ability of TIMP-1 to inhibit protease activity is abrogated, further demonstrating the role of such inhibition in the promotion of synaptic plasticity under well-defined conditions. We also show that dendritic spine maturation involves an intermediate formation of elongated spines, followed by their conversion into mushroom shape. The formation of mushroom-shaped spines is accompanied by increase in AMPA/NMDA ratio of glutamate receptors. Altogether, our results identify inhibition of protease activity as a critical regulatory mechanism for dendritic spines maturation. PMID:27282248

  15. Proteases and Peptidases of Castor Bean Endosperm

    PubMed Central

    Tully, Raymond E.; Beevers, Harry

    1978-01-01

    The endosperm of castor bean seeds (Ricinus communis L.) contains two —SH-dependent aminopeptidases, one hydrolyzing l-leucine-β-naphthylamide optimally at pH 7.0, and the other hydrolyzing l-proline-β-naphthylamide optimally at pH 7.5. After germination the endosperm contains in addition an —SH-dependent hemoglobin protease, a serine-dependent carboxypeptidase, and at least two —SH-dependent enzymes hydrolyzing the model substrate α-N-benzoyl-dl-arginine-β-naphthylamide (BANA). The carboxypeptidase is active on a variety of N-carbobenzoxy dipeptides, especially N-carbobenzoxy-L-phenylalanine-l-alanine and N-carbobenzoxy-l-tyrosine-l-leucine. The pH optima for the protease, carboxypeptidase, and BANAase acivities are 3.5 to 4.0, 5.0 to 5.5, and 6 to 8, respectively. The two aminopeptidases increased about 4-fold in activity during the first 4 days of growth, concurrent with the period of rapid depletion of storage protein. Activities then declined as the endosperm senesced, but were still evident after 6 days. Senescence was complete by day 7 to 8. Hemoglobin protease, carboxypeptidase, and BANAase activities appeared in the endosperm at day 2 to 3, and reached peak activity at day 5 to 6. The data indicate that the aminopeptidases are involved in the early mobilization of endosperm storage protein, whereas protease, carboxypeptidase, and BANAase may take part in later turnover and/or senescence. PMID:16660598

  16. Observations of Titan 3C-4 Transtage Fragmentation Debris

    NASA Technical Reports Server (NTRS)

    Cowardin, Heather; Seitzer, P.; Abercromby, K.; Barker, E.; Cardona, T.; Krisko, P.; Lederer, S.

    2013-01-01

    The fragmentation of a Titan 3C-4 Transtage (1968-081) on 21 February 1992 is one of only two known break-ups in or near geosynchronous orbit. The original rocket body and 24 pieces of debris are currently being tracked by the US Space Surveillance Network (SSN). The rocket body (SSN# 3432) and several of the original fragments (SSN# 25000, 25001, 30000, and 33511) were observed in survey mode during 2004-2010 using the 0.6-m Michigan Orbital DEbris Survey Telescope (MODEST) in Chile using a broad R filter. This paper will present a size distribution for all calibrated magnitude data acquired on MODEST. Size distribution plots will also be shown using historical models for small fragmentation debris (down to 10 cm) believed to be associated with the Titan break-up. In November 2010, visible broadband photometry (Johnson/Kron-Cousins BVRI) was acquired with the 0.9-m Small and Moderate Aperture Research Telescope System (SMARTS) at the Cerro Tololo Inter-American Observatory (CTIO) in Chile on several Titan fragments (SSN# 25001, 33509, 33510) and the parent rocket body. Color index data will be used to determine the fragment brightness distribution and how the data compares to spacecraft materials measured in the laboratory using similar photometric measurement techniques. In 2012, the SSN added 16 additional fragments to the catalogue. MODEST acquired magnitude data on ten Titan fragments in late 2012 and early 2013. The magnitude distribution of all the observed fragments are analyzed as a function of time. In order to better characterize the breakup fragments spectral measurements were acquired on the original rocket body and five Titan fragments using the 6.5-m Magellan telescopes at Las Campanas Observatory in Chile. The telescopic spectra are compared with laboratory acquired spectra of materials (e.g., Aluminum and various paints) and categorized based on known absorption features for spacecraft materials.

  17. Multispectral Imaging of the Jet in 3C346

    NASA Astrophysics Data System (ADS)

    Smith, E. P.; Gardner, J. P.; Heap, S. R.; Baum, S. A.; O'Dea, C. P.

    1998-12-01

    Much progress has been made towards understanding the physics of extra-galactic jet flow in the past 10 years, through numerical modeling and detailed multi-frequency radio mapping. However, we still admit that basic parameters such as the magnetic field strength, the particle density, the bulk Lorentz factor and jet configuration (e.g., fast inner jet and slower outer sheath), and the nature and need for shocks remain unknown. However, by resolving the jets spectrally and spatially and pushing the observations to the higher energies with sufficient spatial resolution to resolve the cooling scale lengths, we can place unique constraints on the physical composition of, and acceleration mechanisms in, extra-galactic jets. The tightest constraints on the physics of jets come not from radio observations, but from observations in the optical, UV, and x-ray, where the lifetimes of the synchrotron emitting particles are measured in hundreds of years and the particles travel distances of hundreds of parsecs. To this end, we present radio through vacuum ultraviolet (STIS FUV-MAMA) spatially resolved images of the radio jet in 3C346 and use them examine the spatial variation of the spectral indices. Because the lifetime of the synchrotron radiating particles is inversely proportional to the observed frequency we can use the vastly different timescales (and therefore length scales) over which the radio (t ~ 10(5-7) yr.) and ultraviolet (t ~ 10(2-3) yr.) emitting particles cool to begin to place constraints on the magnetic field strengths. Also, the curvature of the spectrum as a function of position can be compared with aging models to constrain the magnetic field strength and to test the hypothesis that the magnetic field in radio jets may be substantially below equipartition (Heinz and Begelman 1997).

  18. Multiwavelength Observations of 3C66A in 2003

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed Central

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

    2015-01-01

    Diabetic nephropathy (DN) is one of the major complications of diabetes mellitus (DM), leads to chronic kidney disease (CKD), and, ultimately, is the main cause for end-stage kidney disease (ESKD). Beyond urinary albumin, no reliable biomarkers are available for accurate early diagnostics. Urinary extracellular vesicles (UEVs) have recently emerged as an interesting source of diagnostic and prognostic disease biomarkers. Here we used a protease and respective protease inhibitor array to profile urines of type 1 diabetes patients at different stages of kidney involvement. Urine samples were divided into groups based on the level of albuminuria and UEVs isolated by hydrostatic dialysis and screened for relative changes of 34 different proteases and 32 protease inhibitors, respectively. Interestingly, myeloblastin and its natural inhibitor elafin showed an increase in the normo- and microalbuminuric groups. Similarly, a characteristic pattern was observed in the array of protease inhibitors, with a marked increase of cystatin B, natural inhibitor of cathepsins L, H, and B as well as of neutrophil gelatinase-associated Lipocalin (NGAL) in the normoalbuminuric group. This study shows for the first time the distinctive alterations in comprehensive protease profiles of UEVs in diabetic nephropathy and uncovers intriguing mechanistic, prognostic, and diagnostic features of kidney damage in diabetes. PMID:25874235

  20. Protease IV, a quorum sensing-dependent protease of Pseudomonas aeruginosa modulates insect innate immunity.

    PubMed

    Park, Su-Jin; Kim, Soo-Kyoung; So, Yong-In; Park, Ha-Young; Li, Xi-Hui; Yeom, Doo Hwan; Lee, Mi-Nan; Lee, Bok-Luel; Lee, Joon-Hee

    2014-12-01

    In Pseudomonas aeruginosa, quorum sensing (QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, Tenebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P. aeruginosa functions as a key virulence effector causing the melanization and death of T. molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme (SPE) and SPE-activating enzyme (SAE) without the activation of the antimicrobial peptide (AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T. molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted Tenebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P. aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs. PMID:25315216

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

    SciTech Connect

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

    2009-06-05

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

  2. Coagulation factor XII protease domain crystal structure

    PubMed Central

    Pathak, M; Wilmann, P; Awford, J; Li, C; Hamad, BK; Fischer, PM; Dreveny, I; Dekker, LV; Emsley, J

    2015-01-01

    Background Coagulation factor XII is a serine protease that is important for kinin generation and blood coagulation, cleaving the substrates plasma kallikrein and FXI. Objective To investigate FXII zymogen activation and substrate recognition by determining the crystal structure of the FXII protease domain. Methods and results A series of recombinant FXII protease constructs were characterized by measurement of cleavage of chromogenic peptide and plasma kallikrein protein substrates. This revealed that the FXII protease construct spanning the light chain has unexpectedly weak proteolytic activity compared to β-FXIIa, which has an additional nine amino acid remnant of the heavy chain present. Consistent with these data, the crystal structure of the light chain protease reveals a zymogen conformation for active site residues Gly193 and Ser195, where the oxyanion hole is absent. The Asp194 side chain salt bridge to Arg73 constitutes an atypical conformation of the 70-loop. In one crystal form, the S1 pocket loops are partially flexible, which is typical of a zymogen. In a second crystal form of the deglycosylated light chain, the S1 pocket loops are ordered, and a short α-helix in the 180-loop of the structure results in an enlarged and distorted S1 pocket with a buried conformation of Asp189, which is critical for P1 Arg substrate recognition. The FXII structures define patches of negative charge surrounding the active site cleft that may be critical for interactions with inhibitors and substrates. Conclusions These data provide the first structural basis for understanding FXII substrate recognition and zymogen activation. PMID:25604127

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

    PubMed Central

    Dautin, Nathalie

    2010-01-01

    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. PMID:22069633

  4. Proteases from the Regenerating Gut of the Holothurian Eupentacta fraudatrix

    PubMed Central

    Lamash, Nina E.; Dolmatov, Igor Yu

    2013-01-01

    Four proteases with molecular masses of 132, 58, 53, and 47 kDa were detected in the digestive system of the holothurian Eupentacta fraudatrix. These proteases displayed the gelatinase activity and characteristics of zinc metalloproteinases. The 58 kDa protease had similar protease inhibitor sensitivity to that of mammalian matrix metalloproteinases. Zymographic assay revealed different lytic activities of all four proteases during intestine regeneration in the holothurian. The 132 kDa protease showed the highest activity at the first stage. During morphogenesis (stages 2–4 of regeneration), the highest activity was measured for the 53 and 58 kDa proteases. Inhibition of protease activity exerts a marked effect on regeneration, which was dependent on the time when 1,10-phenanthroline injections commenced. When metalloproteinases were inhibited at the second stage of regeneration, the restoration rates were decreased. However, such an effect proved to be reversible, and when inhibition ceased, the previous rate of regeneration was recovered. When protease activity is inhibited at the first stage, regeneration is completely abolished, and the animals die, suggesting that early activation of the proteases is crucial for triggering the regenerative process in holothurians. The role of the detected proteases in the regeneration processes of holothurians is discussed. PMID:23505505

  5. New directions for protease inhibitors directed drug discovery.

    PubMed

    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. Simultaneous Multiwavelength Monitoring of 3C66A

    NASA Technical Reports Server (NTRS)

    Boettcher, M.

    2004-01-01

    The radio-selected BL Lac object 3C66A was the target of an intensive multiwavelength campaign from Sept. 2003 through Feb. 2004. It was monitored by the Whole Earth Blazar Telescope (WEBT) collaboration, 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 observations using the VLBA are being carried out during the campaign and throughout the year 2004 to follow possible structural changes of the source. 21 pointings with RXTE during the period Sept. 15 - Dec. 27, 2003. All collected data have been fully analyzed, and first results have already been published at the 8th HEAD Meeting in New Orleans, LA, in Sept. 2004, and will also be presented at the 205th AAS Meeting in San Diego, CA, in Jan. 2005. A first Journal paper, to be submitted to the Astrophysical Journal, is currently in preparation, and we plan to have it ready for submission in January 2005. 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 gradual spectral softening in the optical over the entire duration of multi-day outbursts (in both the rising and decaying phase). The X-ray spectrum is consistent with a power-law with a photon spectral index of approx. 2.1, indicating that the RXTE energy band might be located right at the intersection of the synchrotron and the high-energy emission components. No significant flux or spectral variability at X-ray energies was detected, though there seems to be a trend of very modest brightening in tandem with the optical flux. The first 4 VLBA epochs indicate a rather smooth jet with

  7. Hepatitis C virus NS3 protease is activated by low concentrations of protease inhibitors.

    PubMed

    Dahl, Göran; Arenas, Omar Gutiérrez; Danielson, U Helena

    2009-12-01

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a bifunctional enzyme with a protease and a helicase functionality located in each of the two domains of the single peptide chain. There is little experimental evidence for a functional role of this unexpected arrangement since artificial single domain forms of both enzymes are catalytically competent. We have observed that low concentrations of certain protease inhibitors activate the protease of full-length NS3 from HCV genotype 1a with up to 100%, depending on the preincubation time and the inhibitor used. The activation was reduced, but not eliminated, by increased ionic strength, lowered glycerol concentration, or lowered pH. In all cases, it was at the expense of a significant loss of activity. Activation was not seen with the artificial protease domain of genotype 1b NS3 fused with a fragment of the NS4A cofactor. This truncated and covalently modified enzyme form was much less active and exhibited fundamentally different catalytic properties to the full-length NS3 protease without the fused cofactor. The most plausible explanation for the activation was found to involve a slow transition between two enzyme conformations, which differed in their catalytic ability and affinity for inhibitors. Equations derived based on this assumption resulted in better fits to the experimental data than the equation for simple competitive inhibition. The mechanism may involve an inhibitor-induced stabilization of the helicase domain in a conformation that enhances the protease activity, or an improved alignment of the catalytic triad in the protease. The proposed mnemonic mechanism and derived equations are viable for both these explanations and can serve as a basic framework for future studies of enzymes activated by inhibitors or other ligands.

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

    PubMed

    Kousted, Tina M; Skjoedt, Karsten; Petersen, Steen V; Koch, Claus; Vitved, Lars; Sochalska, Maja; Lacroix, Céline; Andersen, Lisbeth M; Wind, Troels; Andreasen, Peter A; Jensen, Jan K

    2014-01-01

    Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all 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 the loop connecting α-helix F with β-strand 3A and the loop connecting α-helix A with β-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1's protease inhibitory activity.

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

    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. PMID:26143919

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

    PubMed

    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.

    Code of Federal Regulations, 2010 CFR

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

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 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...

  14. 40 CFR 152.112 - Approval of registration under FIFRA sec. 3(c)(5).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... sec. 3(c)(5). 152.112 Section 152.112 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... Applications § 152.112 Approval of registration under FIFRA sec. 3(c)(5). EPA will approve an application under the criteria of FIFRA sec. 3(c)(5) only if: (a) The Agency has determined that the application...

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

    PubMed

    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. Structural determinants of tobacco vein mottling virus protease substrate specificity

    SciTech Connect

    Sun, Ping; Austin, Brian P.; Tozer, Jozsef; Waugh, David

    2010-10-28

    Tobacco vein mottling virus (TVMV) is a member of the Potyviridae, one of the largest families of plant viruses. The TVMV genome is translated into a single large polyprotein that is subsequently processed by three virally encoded proteases. Seven of the nine cleavage events are carried out by the NIa protease. Its homolog from the tobacco etch virus (TEV) is a widely used reagent for the removal of affinity tags from recombinant proteins. Although TVMV protease is a close relative of TEV protease, they exhibit distinct sequence specificities. We report here the crystal structure of a catalytically inactive mutant TVMV protease (K65A/K67A/C151A) in complex with a canonical peptide substrate (Ac-RETVRFQSD) at 1.7-{angstrom} resolution. As observed in several crystal structures of TEV protease, the C-terminus ({approx}20 residues) of TVMV protease is disordered. Unexpectedly, although deleting the disordered residues from TEV protease reduces its catalytic activity by {approx}10-fold, an analogous truncation mutant of TVMV protease is significantly more active. Comparison of the structures of TEV and TVMV protease in complex with their respective canonical substrate peptides reveals that the S3 and S4 pockets are mainly responsible for the differing substrate specificities. The structure of TVMV protease suggests that it is less tolerant of variation at the P1{prime} position than TEV protease. This conjecture was confirmed experimentally by determining kinetic parameters k{sub cat} and K{sub m} for a series of oligopeptide substrates. Also, as predicted by the cocrystal structure, we confirm that substitutions in the P6 position are more readily tolerated by TVMV than TEV protease.

  17. Mitochondrial Proteases as Emerging Pharmacological Targets.

    PubMed

    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

  18. Oligomeric state study of prokaryotic rhomboid proteases.

    PubMed

    Sampathkumar, Padmapriya; Mak, Michelle W; Fischer-Witholt, Sarah J; Guigard, Emmanuel; Kay, Cyril M; Lemieux, M Joanne

    2012-12-01

    Rhomboid peptidases (proteases) play key roles in signaling events at the membrane bilayer. Understanding the regulation of rhomboid function is crucial for insight into its mechanism of action. Here we examine the oligomeric state of three different rhomboid proteases. We subjected Haemophilus influenzae, (hiGlpG), Escherichia coli GlpG (ecGlpG) and Bacillus subtilis (YqgP) to sedimentation equilibrium analysis in detergent-solubilized dodecylmaltoside (DDM) solution. For hiGlpG and ecGlpG, rhomboids consisting of the core 6 transmembrane domains without and with soluble domains respectively, and YqgP, predicted to have 7 transmembrane domains with larger soluble domains at the termini, the predominant species was dimeric with low amounts of monomer and tetramers observed. To examine the effect of the membrane domain alone on oligomeric state of rhomboid, hiGlpG, the simplest form from the rhomboid class of intramembrane proteases representing the canonical rhomboid core of six transmembrane domains, was studied further. Using gel filtration and crosslinking we demonstrate that hiGlpG is dimeric and functional in DDM detergent solution. More importantly co-immunoprecipitation studies demonstrate that the dimer is present in the lipid bilayer suggesting a physiological dimer. Overall these results indicate that rhomboids form oligomers which are facilitated by the membrane domain. For hiGlpG we have shown that these oligomers exist in the lipid bilayer. This is the first detailed oligomeric state characterization of the rhomboid family of peptidases. PMID:22921757

  19. Corruption of innate immunity by bacterial proteases.

    PubMed

    Potempa, Jan; Pike, Robert N

    2009-01-01

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

  20. Four Amino Acid Changes in HIV-2 Protease Confer Class-Wide Sensitivity to Protease Inhibitors

    PubMed Central

    Smith, Robert A.; Gottlieb, Geoffrey S.

    2015-01-01

    ABSTRACT Protease is essential for retroviral replication, and protease inhibitors (PI) are important for treating HIV infection. HIV-2 exhibits intrinsic resistance to most FDA-approved HIV-1 PI, retaining clinically useful susceptibility only to lopinavir, darunavir, and saquinavir. The mechanisms for this resistance are unclear; although HIV-1 and HIV-2 proteases share just 38 to 49% sequence identity, all critical structural features of proteases are conserved. Structural studies have implicated four amino acids in the ligand-binding pocket (positions 32, 47, 76, and 82). We constructed HIV-2ROD9 molecular clones encoding the corresponding wild-type HIV-1 amino acids (I32V, V47I, M76L, and I82V) either individually or together (clone PRΔ4) and compared the phenotypic sensitivities (50% effective concentration [EC50]) of mutant and wild-type viruses to nine FDA-approved PI. Single amino acid replacements I32V, V47I, and M76L increased the susceptibility of HIV-2 to multiple PI, but no single change conferred class-wide sensitivity. In contrast, clone PRΔ4 showed PI susceptibility equivalent to or greater than that of HIV-1 for all PI. We also compared crystallographic structures of wild-type HIV-1 and HIV-2 proteases complexed with amprenavir and darunavir to models of the PRΔ4 enzyme. These models suggest that the amprenavir sensitivity of PRΔ4 is attributable to stabilizing enzyme-inhibitor interactions in the P2 and P2′ pockets of the protease dimer. Together, our results show that the combination of four amino acid changes in HIV-2 protease confer a pattern of PI susceptibility comparable to that of HIV-1, providing a structural rationale for intrinsic HIV-2 PI resistance and resolving long-standing questions regarding the determinants of differential PI susceptibility in HIV-1 and HIV-2. IMPORTANCE Proteases are essential for retroviral replication, and HIV-1 and HIV-2 proteases share a great deal of structural similarity. However, only three of nine

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

    PubMed

    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.

  2. New Potent SARS-CoV 3C-Like Protease Inhibitors Derived from Thieno[2,3-d]-pyrimidine Derivatives.

    PubMed

    Abd El-All, Amira S; Atta, Sanaa M Sh; Roaiah, Hanaa M F; Awad, Enas M; Abdalla, Mohamed M

    2016-03-01

    2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxamide (1) condensed with carbaldehydes 2a,b to give the respective thienopyrimidines (3a,b), which reacted with phosphoryl chloride and hydrazine hydrate to afford the respective pyrimidinohydrazines (4a,b). Compound 4a condensed with acetophenone under Vilsmeier conditions to afford the formylated pyrazolopyrimidine 6. Condensation of 4a with active methylenes produced the respective pyrazolopyrimidines (7-11). Besides, 4a condensed with succinic anhydride and with phthalic anhydride, yielding the pyrrolidine-2,5-dione 12 and the isoindoline-1,3-dione 13, respectively. Moreover, 4a reacted with isatin to afford the hydrazono-indolin-2-one 14. Structural elucidations for the new thienopyrimidines were based upon compatible analytical and spectroscopic results. Eleven of the new compounds were tested and found active against influenza A neuraminidase virus (H3N2). Compounds 12 and 13 were the most potent. PMID:26806115

  3. Multifrequency VLBI follow up study of strong γ-ray flares in the blazars 3C273 and 3C279

    NASA Astrophysics Data System (ADS)

    Lisakov, Mikhail M.; Kovalev, Yuri Y.

    2015-03-01

    We present multifrequency VLBI observations of the blazars 3C273 and 3C279 after detecting strong γ-ray flares in both of them. 3C273 exhibited a prominent flare in γ-rays in September 2009 which was followed by a strong flare in the 7 mm VLBI core and emergence of a new feature in the parsec scale jet. We have used time delay between flares in different wavebands together with kinematic analysis to determine that the γ-ray emission zone in 3C273 is located 3.6-5.3 pc upstream from the apparent 7 mm core. We have also analyzed frequency dependent core position to measure a deprojected distance between 7 mm core and the true base of the jet: 1-6 pc for 3C273 and 1-3 pc for 3C279, depending on observing epoch. For 3C279 light curve analysis did not give a robust γ-radio delay because there were too many overlapping flares in this source during considered period.

  4. Diversity of Both the Cultivable Protease-Producing Bacteria and Bacterial Extracellular Proteases in the Coastal Sediments of King George Island, Antarctica

    PubMed Central

    Zhou, Ming-Yang; Wang, Guang-Long; Li, Dan; Zhao, Dian-Li; Qin, Qi-Long; Chen, Xiu-Lan; Chen, Bo; Zhou, Bai-Cheng; Zhang, Xi-Ying; Zhang, Yu-Zhong

    2013-01-01

    Protease-producing bacteria play a vital role in degrading sedimentary organic nitrogen. However, the diversity of these bacteria and their extracellular proteases in most regions remain unknown. In this paper, the diversity of the cultivable protease-producing bacteria and of bacterial extracellular proteases in the sediments of Maxwell Bay, King George Island, Antarctica was investigated. The cultivable protease-producing bacteria reached 105 cells/g in all 8 sediment samples. The cultivated protease-producing bacteria were mainly affiliated with the phyla Actinobacteria, Firmicutes, Bacteroidetes, and Proteobacteria, and the predominant genera were Bacillus (22.9%), Flavobacterium (21.0%) and Lacinutrix (16.2%). Among these strains, Pseudoalteromonas and Flavobacteria showed relatively high protease production. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria were serine proteases or metalloproteases. These results begin to address the diversity of protease-producing bacteria and bacterial extracellular proteases in the sediments of the Antarctic Sea. PMID:24223990

  5. Epstein-Barr virus nuclear antigen 3C regulated genes in lymphoblastoid cell lines.

    PubMed

    Zhao, Bo; Mar, Jessica C; Maruo, Seiji; Lee, Sungwook; Gewurz, Benjamin E; Johannsen, Eric; Holton, Kristina; Rubio, Renee; Takada, Kenzo; Quackenbush, John; Kieff, Elliott

    2011-01-01

    EBV nuclear antigen 3C (EBNA3C) is an essential transcription factor for EBV transformed lymphoblast cell line (LCL) growth. To identify EBNA3C-regulated genes in LCLs, microarrays were used to measure RNA abundances in each of three different LCLs that conditionally express EBNA3C fused to a 4-OH-Tamoxifen-dependent estrogen receptor hormone binding domain (EBNA3CHT). At least three RNAs were assayed for each EBNA3CHT LCL under nonpermissive conditions, permissive conditions, and nonpermissive conditions with wild-type EBNA3C transcomplementation. Using a two-way ANOVA model of EBNA3C levels, we identified 550 regulated genes that were at least 1.5-fold up- or down-regulated with false discovery rates < 0.01. EBNA3C-regulated genes overlapped significantly with genes regulated by EBNA2 and EBNA3A consistent with coordinated effects on cell gene transcription. Of the 550 EBNA3C-regulated genes, 106 could be placed in protein networks. A seeded Bayesian network analysis of the 80 most significant EBNA3C-regulated genes suggests that RAC1, LYN, and TNF are upstream of other EBNA3C-regulated genes. Gene set enrichment analysis found enrichment for MAP kinase signaling, cytokine-cytokine receptor interactions, JAK-STAT signaling, and cell adhesion molecules, implicating these pathways in EBNA3C effects on LCL growth or survival. EBNA3C significantly up-regulated the CXCL12 ligand and its CXCR4 receptor and increased LCL migration. CXCL12 up-regulation depended on EBNA3C's interaction with the cell transcription factor, RBPJ, which is essential for LCL growth. EBNA3C also up-regulated MYC 1.3-fold and down-regulated CDKN2A exons 2 and 3, shared by p16 and p14, 1.4-fold, with false discovery rates < 5 × 10(-4).

  6. Economic Methods of Ginger Protease'sextraction and Purification

    NASA Astrophysics Data System (ADS)

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

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

  7. A preliminary neutron diffraction analysis of Achromobacter protease I

    NASA Astrophysics Data System (ADS)

    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. Role of Protease-Inhibitors in Ocular Diseases.

    PubMed

    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

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

    SciTech Connect

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

    2011-09-01

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

  10. Extracellular Bacterial Proteases in Chronic Wounds: A Potential Therapeutic Target?

    PubMed Central

    Suleman, Louise

    2016-01-01

    Significance: Bacterial biofilms are considered to be responsible for over 80% of persistent infections, including chronic lung infections, osteomyelitis, periodontitis, endocarditis, and chronic wounds. Over 60% of chronic wounds are colonized with bacteria that reside within a biofilm. The exaggerated proteolytic environment of chronic wounds, more specifically elevated matrix metalloproteinases, is thought to be one of the possible reasons as to why chronic wounds fail to heal. However, the role of bacterial proteases within chronic wounds is not fully understood. Recent Advances: Recent research has shown that bacterial proteases can enable colonization and facilitate bacterial immune evasion. The inhibition of bacterial proteases such as Pseudomonas aeruginosa elastase B (LasB) has resulted in the disruption of the bacterial biofilm in vitro. P. aeruginosa is thought to be a key pathogen in chronic wound infection, and therefore, the disruption of these biofilms, potentially through the targeting of P. aeruginosa bacterial proteases, is an attractive therapeutic endeavor. Critical Issues: Disrupting biofilm formation through the inhibition of bacterial proteases may lead to the dissemination of bacteria from the biofilm, allowing planktonic cells to colonize new sites within the wound. Future Directions: Despite a plethora of evidence supporting the role of bacterial proteases as virulence factors in infection, there remains a distinct lack of research into the effect of bacterial proteases in chronic wounds. To assess the viability of targeting bacterial proteases, future research should aim to understand the role of these proteases in a variety of chronic wound subtypes. PMID:27785379

  11. Role of Protease-Inhibitors in Ocular Diseases.

    PubMed

    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.

  12. Detergent alkaline proteases: enzymatic properties, genes, and crystal structures.

    PubMed

    Saeki, Katsuhisa; Ozaki, Katsuya; Kobayashi, Tohru; Ito, Susumu

    2007-06-01

    Subtilisin-like serine proteases from bacilli have been used in various industrial fields worldwide, particularly in the production of laundry and automatic dishwashing detergents. They belong to family A of the subtilase superfamily, which is composed of three clans, namely, true subtilisins, high-alkaline proteases, and intracellular proteases. We succeeded in the large-scale production of a high-alkaline protease (M-protease) from alkaliphilic Bacillus clausii KSM-K16, and the enzyme has been introduced into compact heavy-duty laundry detergents. We have also succeeded in the industrial-scale production of a new alkaline protease, KP-43, which was originally resistant to chemical oxidants and to surfactants, produced by alkaliphilic Bacillus sp. strain KSM-KP43 and have incorporated it into laundry detergents. KP-43 and related proteases form a new clan, oxidatively stable proteases, in subtilase family A. In this review, we describe the enzymatic properties, gene sequences, and crystal structures of M-protease, KP-43, and related enzymes. PMID:17630120

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

    PubMed

    Kocabiyik, Semra; Erdem, Bilge

    2002-08-01

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

  14. CFD Growth of 3C-SiC on 4H/6H Mesas

    NASA Technical Reports Server (NTRS)

    Neudeck, Philip G.; Trunek, Andrew J.; Spry, David J.; Powell, J. Anthony; Du, Hui; Skowronski, Marek; Huang, XianRong; Dudley, Michael

    2006-01-01

    This article describes growth and characterization of the highest quality reproducible 3C-SiC heteroepitaxial films ever reported. By properly nucleating 3C-SiC growth on top of perfectly on-axis (0001) 4H-SiC mesa surfaces completely free of atomic scale steps and extended defects, growth of 3C-SiC mesa heterofilms completely free of extended crystal defects can be achieved. In contrast, nucleation and growth of 3C-SiC mesa heterofilms on top of 4H-SiC mesas with atomic-scale steps always results in numerous observable dislocations threading through the 3C-SiC epilayer. High-resolution X-ray diffraction and transmission electron microscopy measurements indicate non-trivial in-plane lattice mismatch between the 3C and 4H layers. This mismatch is somewhat relieved in the step-free mesa case via misfit dislocations confined to the 3C/4H interfacial region without dislocations threading into the overlying 3C-SiC layer. These results indicate that the presence or absence of steps at the 3C/4H heteroepitaxial interface critically impacts the quality, defect structure, and relaxation mechanisms of single-crystal heteroepitaxial 3C-SiC films.

  15. CVD Growth of 3C-SiC on 4H/6H Mesas

    SciTech Connect

    Neudeck,P.; Trunek, A.; Spry, D.; Powell, J.; Du, H.; Skowronski, M.; Huang, X.; Dudley, M.

    2006-01-01

    This article describes growth and characterization of the highest quality reproducible 3C-SiC heteroepitaxial films ever reported. By properly nucleating 3C-SiC growth on top of perfectly on-axis (0001) 4H-SiC mesa surfaces completely free of atomic scale steps and extended defects, growth of 3C-SiC mesa heterofilms completely free of extended crystal defects can be achieved. In contrast, nucleation and growth of 3C-SiC mesa heterofilms on top of 4H-SiC mesas with atomic-scale steps always results in numerous observable dislocations threading through the 3C-SiC epilayer. High-resolution X-ray diffraction (HRXRD) and high resolution cross-sectional transmission electron microscopy (HRXTEM) measurements indicate non-trivial, in-plane, lattice mismatch between the 3C and 4H layers. This mismatch is somewhat relieved in the step-free mesa case via misfit dislocations confined to the 3C/4H interfacial region without dislocations threading into the overlying 3C-SiC layer. These results indicate that the presence or absence of steps at the 3C/4H heteroepitaxial interface critically impacts the quality, defect structure, and relaxation mechanisms of single-crystal heteroepitaxial 3C-SiC films.

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

    PubMed

    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

  17. Origin and Diversification of Meprin Proteases.

    PubMed

    Marín, Ignacio

    2015-01-01

    Meprins are astacin metalloproteases with a characteristic, easily recognizable structure, given that they are the only proteases with both MAM and MATH domains plus a transmembrane region. So far assumed to be vertebrate-specific, it is shown here, using a combination of evolutionary and genomic analyses, that meprins originated before the urochordates/vertebrates split. In particular, three genes encoding structurally typical meprin proteins are arranged in tandem in the genome of the urochordate Ciona intestinalis. Phylogenetic analyses showed that the protease and MATH domains present in the meprin-like proteins encoded by the Ciona genes are very similar in sequence to the domains found in vertebrate meprins, which supports them having a common origin. While many vertebrates have the two canonical meprin-encoding genes orthologous to human MEP1A and MEP1B (which respectively encode for the proteins known as meprin α and meprin β), a single gene has been found so far in the genome of the chondrichthyan fish Callorhinchus milii, and additional meprin-encoding genes are present in some species. Particularly, a group of bony fish species have genes encoding highly divergent meprins, here named meprin-F. Genes encoding meprin-F proteins, derived from MEP1B genes, are abundant in some species, as the Amazon molly, Poecilia formosa, which has 7 of them. Finally, it is confirmed that the MATH domains of meprins are very similar to the ones in TRAF ubiquitin ligases, which suggests that meprins originated when protease and TRAF E3-encoding sequences were combined. PMID:26288188

  18. Purification of Pseudomonas aeruginosa proteases and microscopic characterization of pseudomonal protease-induced rabbit corneal damage.

    PubMed Central

    Kreger, A S; Gray, L D

    1978-01-01

    Extracellular proteases of three cornea-virulent strains of Pseudomonas aeruginosa were isolated by sequential ammonium sulfate precipitation, Ultrogel AcA 54 gel filtration, and flat-bed isoelectric focusing. The purity of the preparations was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis , thin-layer isoelectric focusing in polyacrylamide gel, immunodiffusion and immunoelectrophoretic procedures, and tests for the presence of other known pseudomonal products. Light and electron microscopic examination of rabbit corneal lesions observed 4 to 6 h after the intracorneal injection of submicrogram amounts of the proteases revealed: (i) degeneration and necrosis of epithelium, endothelium, and keratocytes, (ii) infiltration, degeneration, and necrosis of polymorphonuclear leukocytes, (iii) loss of the characteristic weblike pattern, colloidal iron staining, and ruthenium red staining of the stromal proteoglycan ground substance, (iv) dispersal of strucutrally normal appearing collagen fibrils, ground substance, (iv) dispersal of structurally normal appearing collagen fibrils, and (v) accumulation of plasma proteins and fibrin in the necrotic corneas. These structural alterations are very similar to those observed previously during experimental P. aeruginosa keratitis, and this similarity supports the idea that pseudomonal proteases are responsible, at least in part, for the rapid and extensive liquefaction necrosis characteristic of pseudomonal-induced keratitis. In addition, the results support the idea that pseudomonal proteases elicit severe corneal damage by causing the loss of the corneal proteoglycan ground substance, thus resulting in dispersal of undamaged collagen fibrils, weakening of the corneal stroma, and subsequent descemetocele formation and corneal perforation by the anterior chamber pressure. Images PMID:415981

  19. A bumblebee (Bombus ignitus) venom serine protease inhibitor that acts as a microbial serine protease inhibitor.

    PubMed

    Wan, Hu; Kim, Bo Yeon; Lee, Kwang Sik; Yoon, Hyung Joo; Lee, Kyung Yong; Jin, Byung Rae

    2014-01-01

    Serine protease inhibitors from bumblebee venom have been shown to block plasmin activity. In this study, we identified the protein BiVSPI from the venom of Bombus ignitus to be a serine protease inhibitor and an antimicrobial factor. BiVSPI is a 55-amino acid mature peptide with ten conserved cysteine residues and a P1 methionine residue. BiVSPI is expressed in the venom gland and also found in the venom as an 8-kDa peptide. Recombinant BiVSPI that was expressed in baculovirus-infected insect cells exhibited inhibitory activity against chymotrypsin but not trypsin. BiVSPI also inhibited microbial serine proteases, such as subtilisin A (Ki=6.57nM) and proteinase K (Ki=7.11nM). In addition, BiVSPI was shown to bind directly to Bacillus subtilis, Bacillus thuringiensis, and Beauveria bassiana but not to Escherichia coli. Consistent with these results, BiVSPI exhibited antimicrobial activity against Gram-positive bacteria and fungi. These findings provide evidence for a novel serine protease inhibitor in bumblebee venom that has antimicrobial functions.

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

    SciTech Connect

    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

    2015-06-30

    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.

  1. Rigidity analysis of HIV-1 protease

    NASA Astrophysics Data System (ADS)

    Heal, J. W.; Wells, S. A.; Jimenez-Roldan, E.; Freedman, R. F.; Römer, R. A.

    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 β-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.

  2. Endogenous Protease Activation of ENaC

    PubMed Central

    Adebamiro, Adedotun; Cheng, Yi; Johnson, John P.; Bridges, Robert J.

    2005-01-01

    Endogenous serine proteases have been reported to control the reabsorption of Na+ by kidney- and lung-derived epithelial cells via stimulation of electrogenic Na+ transport mediated by the epithelial Na+ channel (ENaC). In this study we investigated the effects of aprotinin on ENaC single channel properties using transepithelial fluctuation analysis in the amphibian kidney epithelium, A6. Aprotinin caused a time- and concentration-dependent inhibition (84 ± 10.5%) in the amiloride-sensitive sodium transport (INa) with a time constant of 18 min and half maximal inhibition constant of 1 μM. Analysis of amiloride analogue blocker–induced fluctuations in INa showed linear rate–concentration plots with identical blocker on and off rates in control and aprotinin-inhibited conditions. Verification of open-block kinetics allowed for the use of a pulse protocol method (Helman, S.I., X. Liu, K. Baldwin, B.L. Blazer-Yost, and W.J. Els. 1998. Am. J. Physiol. 274:C947–C957) to study the same cells under different conditions as well as the reversibility of the aprotinin effect on single channel properties. Aprotinin caused reversible changes in all three single channel properties but only the change in the number of open channels was consistent with the inhibition of INa. A 50% decrease in INa was accompanied by 50% increases in the single channel current and open probability but an 80% decrease in the number of open channels. Washout of aprotinin led to a time-dependent restoration of INa as well as the single channel properties to the control, pre-aprotinin, values. We conclude that protease regulation of INa is mediated by changes in the number of open channels in the apical membrane. The increase in the single channel current caused by protease inhibition can be explained by a hyperpolarization of the apical membrane potential as active Na+ channels are retrieved. The paradoxical increase in channel open probability caused by protease inhibition will require further

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

    PubMed

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

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

  4. Antigens of monoclonal antibody NB3C4 are novel markers for oligodendrocytes.

    PubMed

    Yoshimura, K; Kametani, F; Shimoda, Y; Fujimaki, K; Sakurai, Y; Kitamura, K; Asou, H; Nomura, M

    2001-02-12

    We produced NB3C4, a novel monoclonal antibody specific for oligodendrocytes, using human neuroblastoma IMR-32 cells. NB3C4 specifically recognized oligodendrocytes in the CNS, although it bound to neuroblastoma IMR-32 cells and oligodendrocytes in vitro. Double immunofluorescence staining of rat brain using NB3C4 and anti-GST-pi, anti-glial fibrillary acidic protein (GFAP), or anti-neurofilament 200 (NF) antibody revealed that anti-GST-pi antibody identified an oligodendrocyte marker recognizing NB3C4-positive cells, while both anti-GFAP and anti-NF antibody did not. Western blotting of rat brain homogenates showed that NB3C4 bound three proteins of 22-28 kDa, while the anti-GST-pi recognized a 27 kDa protein. Therefore, antigens recognized by NB3C4 could be novel markers for oligodendrocytes.

  5. 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.

    PubMed

    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

  6. 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

    PubMed Central

    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

  7. 3C-SiC/ZnS heterostructured nanospheres with high photocatalytic activity and enhancement mechanism

    SciTech Connect

    Zhang, J.; Wu, X. L. E-mail: paul.chu@cityu.edu.hk; Liu, L. Z.; Yang, L.; Gan, Z. X.; Chu, Paul K. E-mail: paul.chu@cityu.edu.hk

    2015-03-15

    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.

  8. Synthesis and X-ray studies of novel 3-C-nitromethyl-hexofuranoses.

    PubMed

    Turks, Māris; Vēze, Krista; Kiseļovs, Gļebs; Mackeviča, Jevgeņija; Lugiņina, Jevgeņija; Mishnev, Anatoly; Marković, Dean

    2014-06-01

    A practical method for the synthesis of three novel 3-C-nitromethyl-hexofuranoses is reported. The Henry reaction on a 1,2:5,6-di-O-isopropylidene-α-d-gulofuranose-derived ketone provided a 3-C-branched gulo-isomer as the sole reaction product. The dehydration-rehydration of the latter yielded an isopropylidene-protected 3-C-nitromethyl-galactofuranose. The reaction sequence can be also used for the synthesis of a 3-deoxy-3-C-nitromethyl-hexofuranose derivative with a gulo-configuration. Two of the newly obtained carbohydrate derivatives were characterized by X-ray crystallography.

  9. The nature of the optical variations of Seyfert galaxy 3C 120

    SciTech Connect

    Webb, J.R. Austin State Univ., TX )

    1990-01-01

    Results are presented from 61 years of optical observations of the Seyfert galaxy 3C 120. A previously published model of the 3C 120 light curve, derived from power spectrum analysis, is found to be valid for historical as well as current data. It is concluded that the optical variations of 3C 120 can be separated into a linear component, a sinusoidal component, and rapid, high-amplitude flares. Possible sources of the regular variations observed in 3C 120 are also suggested in the context of accretion models and other theoretical models. 15 refs.

  10. Viral proteases as targets for drug design.

    PubMed

    Skoreński, Marcin; Sieńczyk, Marcin

    2013-01-01

    In order to productively infect a host, viruses must enter the cell and force host cell replication mechanisms to produce new infectious virus particles. The success of this process unfortunately results in disease progression and, in the case of infection with many viral species, may cause mortality. The discoveries of Louis Pasteur and Edward Jenner led to one of the greatest advances in modern medicine - the development of vaccines that generate long-lasting memory immune responses to combat viral infection. Widespread use of vaccines has reduced mortality and morbidity associated with viral infection and, in some cases, has completely eradicated virus from the human population. Unfortunately, several viral species maintain a significant ability to mutate and "escape" vaccine-induced immune responses. Thus, novel anti-viral agents are required for treatment and prevention of viral disease. Targeting proteases that are crucial in the viral life cycle has proven to be an effective method to control viral infection, and this avenue of investigation continues to generate anti-viral treatments. Herein, we provide the reader with a brief history as well as a comprehensive review of the most recent advances in the design and synthesis of viral protease inhibitors. PMID:23016690

  11. Broad-Spectrum Allosteric Inhibition of Herpesvirus Proteases

    PubMed Central

    2015-01-01

    Herpesviruses rely on a homodimeric protease for viral capsid maturation. A small molecule, DD2, previously shown to disrupt dimerization of Kaposi’s sarcoma-associated herpesvirus protease (KSHV Pr) by trapping an inactive monomeric conformation and two analogues generated through carboxylate bioisosteric replacement (compounds 2 and 3) were shown to inhibit the associated proteases of all three human herpesvirus (HHV) subfamilies (α, β, and γ). Inhibition data reveal that compound 2 has potency comparable to or better than that of DD2 against the tested proteases. Nuclear magnetic resonance spectroscopy and a new application of the kinetic analysis developed by Zhang and Poorman [Zhang, Z. Y., Poorman, R. A., et al. (1991) J. Biol. Chem. 266, 15591–15594] show DD2, compound 2, and compound 3 inhibit HHV proteases by dimer disruption. All three compounds bind the dimer interface of other HHV proteases in a manner analogous to binding of DD2 to KSHV protease. The determination and analysis of cocrystal structures of both analogues with the KSHV Pr monomer verify and elaborate on the mode of binding for this chemical scaffold, explaining a newly observed critical structure–activity relationship. These results reveal a prototypical chemical scaffold for broad-spectrum allosteric inhibition of human herpesvirus proteases and an approach for the identification of small molecules that allosterically regulate protein activity by targeting protein–protein interactions. PMID:24977643

  12. Effect of proteases on the. beta. -thromboglobulin radioimmunoassay

    SciTech Connect

    Donlon, J.A.; Helgeson, E.A.; Donlon, M.A.

    1985-02-11

    Rat peritoneal mast cells and mast cell granules were evaluated by radioimmunoassay for the presence of ..beta..-thromboglobulin and platelet factor 4. The initial assays indicated that a ..beta..-thromboglobulin cross reacting material was released from mast cells by compound 48/80 in a similar dose-dependent manner as histamine release. The material was also found to be associated with purified granules. However, the use of protease inhibitors in the buffers completely abolished the positive assays. Further evaluation of the effects of various proteases on the ..beta..-thromboglobulin assay indicated that elastase would also generate a false positive assay which could then be neutralized by the use of ..cap alpha../sub 1/-antitrypsin as a protease inhibitor. There was no protease effect on the platelet factor 4 radioimmunoassay which always showed no detectable amounts with mast cells, granules or proteases. These results clearly indicate the artifactual positive assays which can arise when using certain radioimmunoassay tests in the presence of cell proteases. The use of protease inhibitors is a necessary control when applying a radioimmunoassay to a system with potentially active proteases. 24 references, 2 figures, 4 tables.

  13. Expression and characterization of Coprothermobacter proteolyticus alkaline serine protease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  14. Terminal Interface Conformations Modulate Dimer Stability Prior to Amino Terminal Autoprocessing of HIV-1 Protease

    SciTech Connect

    Agniswamy, Johnson; Sayer, Jane M.; Weber, Irene T.; Louis, John M.

    2012-04-17

    The HIV-1 protease (PR) mediates its own release (autoprocessing) from the polyprotein precursor, Gag-Pol, flanked by the transframe region (TFR) and reverse transcriptase at its N- and C-termini, respectively. Autoprocessing at the N-terminus of PR mediates stable dimer formation essential for catalytic activity, leading to the formation of infectious virus. An antiparallel {beta}-sheet interface formed by the four N- and C-terminal residues of each subunit is important for dimer stability. Here, we present the first high-resolution crystal structures of model protease precursor-clinical inhibitor (PI darunavir or saquinavir) complexes, revealing varying conformations of the N-terminal flanking (S{sup -4}FNF{sup -1}) and interface residues (P{sup 1}QIT{sup 4}). A 180{sup o} rotation of the T{sup 4}-L{sup 5} peptide bond is accompanied by a new Q{sup 2}-L{sup 5} hydrogen bond and complete disengagement of PQIT from the {beta}-sheet dimer interface, which may be a feature for intramolecular autoprocessing. This result is consistent with drastically lower thermal stability by 14-20 C of PI complexes of precursors and the mature PR lacking its PQIT residues (by 18.3 C). Similar to the TFR-PR precursor, this deletion also results in a darunavir dissociation constant (2 x 10{sup 4})-fold higher and a markedly increased dimer dissociation constant relative to the mature PR. The terminal {beta}-sheet perturbations of the dimeric structure likely account for the drastically poorer inhibition of autoprocessing of TFR-PR relative to the mature PR, even though significant differences in active site-PI interactions in these structures were not observed. The novel conformations of the dimer interface may be exploited to target selectively the protease precursor prior to its N-terminal cleavage.

  15. Alkaline protease production by a strain of marine yeasts

    NASA Astrophysics Data System (ADS)

    Ping, Wang; Zhenming, Chi; Chunling, Ma

    2006-07-01

    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°C. The optimal medium for the maximum alkaline protease production of strain 10 was 2.5g soluble starch and 2.0g NaNO3 in 100mL seawater with initial pH 6.0. The optimal cultivation conditions for the maximum protease production were temperature 24.5°C, aeration rate 8.0L min-1 and agitation speed 150r min-1 Under the optimal conditions, 623.1 U mg-1 protein of alkaline protease was reached in the culture within 30h of fermentation.

  16. Purification and characterization of an alkaline protease from Acetes chinensis

    NASA Astrophysics Data System (ADS)

    Xu, Jiachao; Liu, Xin; Li, Zhaojie; Xu, Jie; Xue, Changhu; Gao, Xin

    2005-07-01

    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 17.15 folds 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°C 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.

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

    PubMed

    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.

  18. Gemin5 proteolysis reveals a novel motif to identify L protease targets.

    PubMed

    Piñeiro, David; Ramajo, Jorge; Bradrick, Shelton S; Martínez-Salas, Encarnación

    2012-06-01

    Translation of picornavirus RNA is governed by the internal ribosome entry site (IRES) element, directing the synthesis of a single polyprotein. Processing of the polyprotein is performed by viral proteases that also recognize as substrates host factors. Among these substrates are translation initiation factors and RNA-binding proteins whose cleavage is responsible for inactivation of cellular gene expression. Foot-and-mouth disease virus (FMDV) encodes two proteases, L(pro) and 3C(pro). Widespread definition of L(pro) targets suffers from the lack of a sufficient number of characterized substrates. Here, we report the proteolysis of the IRES-binding protein Gemin5 in FMDV-infected cells, but not in cells infected by other picornaviruses. Proteolysis was specifically associated with expression of L(pro), yielding two stable products, p85 and p57. In silico search of putative L targets within Gemin5 identified two sequences whose potential recognition was in agreement with proteolysis products observed in infected cells. Mutational analysis revealed a novel L(pro) target sequence that included the RKAR motif. Confirming this result, the Fas-ligand Daxx, was proteolysed in FMDV-infected and L(pro)-expressing cells. This protein carries a RRLR motif whose substitution to EELR abrogated L(pro) recognition. Thus, the sequence (R)(R/K)(L/A)(R) defines a novel motif to identify putative targets of L(pro) in host factors.

  19. Positive selection of digestive Cys proteases in herbivorous Coleoptera.

    PubMed

    Vorster, Juan; Rasoolizadeh, Asieh; Goulet, Marie-Claire; Cloutier, Conrad; Sainsbury, Frank; Michaud, Dominique

    2015-10-01

    Positive selection is thought to contribute to the functional diversification of insect-inducible protease inhibitors in plants in response to selective pressures exerted by the digestive proteases of their herbivorous enemies. Here we assessed whether a reciprocal evolutionary process takes place on the insect side, and whether ingestion of a positively selected plant inhibitor may translate into a measurable rebalancing of midgut proteases in vivo. Midgut Cys proteases of herbivorous Coleoptera, including the major pest Colorado potato beetle (Leptinotarsa decemlineata), were first compared using a codon-based evolutionary model to look for the occurrence of hypervariable, positively selected amino acid sites among the tested sequences. Hypervariable sites were found, distributed within -or close to- amino acid regions interacting with Cys-type inhibitors of the plant cystatin protein family. A close examination of L. decemlineata sequences indicated a link between their assignment to protease functional families and amino acid identity at positively selected sites. A function-diversifying role for positive selection was further suggested empirically by in vitro protease assays and a shotgun proteomic analysis of L. decemlineata Cys proteases showing a differential rebalancing of protease functional family complements in larvae fed single variants of a model cystatin mutated at positively selected amino acid sites. These data confirm overall the occurrence of hypervariable, positively selected amino acid sites in herbivorous Coleoptera digestive Cys proteases. They also support the idea of an adaptive role for positive selection, useful to generate functionally diverse proteases in insect herbivores ingesting functionally diverse, rapidly evolving dietary cystatins. PMID:26264818

  20. 17 CFR 270.3c-2 - Definition of beneficial ownership in small business investment companies.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 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 Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT...

  1. 17 CFR 270.3c-2 - Definition of beneficial ownership in small business investment companies.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 17 Commodity and Securities Exchanges 3 2011-04-01 2011-04-01 false Definition of beneficial ownership in small business investment companies. 270.3c-2 Section 270.3c-2 Commodity and Securities Exchanges SECURITIES AND EXCHANGE COMMISSION (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT...

  2. 50 CFR Table 3c to Part 680 - Crab Product Codes for Economic Data Reports

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 50 Wildlife and Fisheries 9 2010-10-01 2010-10-01 false Crab Product Codes for Economic Data... EXCLUSIVE ECONOMIC ZONE OFF ALASKA Pt. 680, Table 3c Table 3c to Part 680—Crab Product Codes for Economic Data Reports Code Description 01 Whole crab. 80 Sections. 81 Meats. 97 Other (specify)....

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

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 50 Wildlife and Fisheries 13 2012-10-01 2012-10-01 false Crab Product Codes for Economic Data... EXCLUSIVE ECONOMIC ZONE OFF ALASKA Pt. 680, Table 3c Table 3c to Part 680—Crab Product Codes for Economic Data Reports Code Description 01 Whole crab. 80 Sections. 81 Meats. 97 Other (specify)....

  4. 50 CFR Table 3c to Part 680 - Crab Product Codes for Economic Data Reports

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... 50 Wildlife and Fisheries 11 2011-10-01 2011-10-01 false Crab Product Codes for Economic Data... EXCLUSIVE ECONOMIC ZONE OFF ALASKA Pt. 680, Table 3c Table 3c to Part 680—Crab Product Codes for Economic Data Reports Code Description 01 Whole crab. 80 Sections. 81 Meats. 97 Other (specify)....

  5. Eukaryotic initiation factor 3C silencing inhibits cell proliferation and promotes apoptosis in human glioma.

    PubMed

    Hao, Jinmin; Wang, Zhiming; Wang, Yaowu; Liang, Zhaohui; Zhang, Xin; Zhao, Zongmao; Jiao, Baohua

    2015-06-01

    Eukaryotic initiation factor 3, subunit c (eIF3c), an oncogene overexpressed in human cancers, plays an important role in cell tumorigenesis and proliferation. However, studies assessing its function in gliomas are scarce. The present study evaluated for the first time, the role of eIF3c in gliomas. Immunohistochemistry was carried out to assess eIF3c expression in 95 human glioma samples and normal brain tissues. Then, the eIF3c mRNA levels were detected in tumor and normal brain specimens by quantitative RT-PCR. In addition, eIF3c mRNA levels were assessed in four glioma cell lines (U87, U251, A172 and U373) by semi-quantitative RT-PCR. The RNA interference (RNAi) technology was employed to knock down the eIF3c gene in the U251 cells. Western blot analysis, BrdU assay and flow cytometry were used to measure eIF3c protein levels, cell proliferation, cell apoptosis and cell cycle, respectively. The eIF3c protein was overexpressed in the human glioma specimens. In agreement, the eIF3c mRNA expression levels were significantly higher in the human glioma tissues compared with the normal brain samples (P<0.0001). In addition, eIF3c mRNA was detected in all the glioma cell lines. Silencing the eIF3c gene in the U251 cells by RNAi significantly suppressed cell proliferation (P<0.01) and increased apoptosis (P<0.01). Finally, a stark decrease was observed in the G1 phase cell number (P<0.01), while the S and G2 phase cells were significantly increased (P<0.01) after eIF3c knockdown. These findings suggest that eIF3c is overexpressed in human gliomas and essential for their proliferation and survival. Therefore, inhibiting eIF3c expression may constitute an effective therapy for human glioma.

  6. Active Site Characterization of Proteases Sequences from Different Species of Aspergillus.

    PubMed

    Morya, V K; Yadav, Virendra K; Yadav, Sangeeta; Yadav, Dinesh

    2016-09-01

    A total of 129 proteases sequences comprising 43 serine proteases, 36 aspartic proteases, 24 cysteine protease, 21 metalloproteases, and 05 neutral proteases from different Aspergillus species were analyzed for the catalytically active site residues using MEROPS database and various bioinformatics tools. Different proteases have predominance of variable active site residues. In case of 24 cysteine proteases of Aspergilli, the predominant active site residues observed were Gln193, Cys199, His364, Asn384 while for 43 serine proteases, the active site residues namely Asp164, His193, Asn284, Ser349 and Asp325, His357, Asn454, Ser519 were frequently observed. The analysis of 21 metalloproteases of Aspergilli revealed Glu298 and Glu388, Tyr476 as predominant active site residues. In general, Aspergilli species-specific active site residues were observed for different types of protease sequences analyzed. The phylogenetic analysis of these 129 proteases sequences revealed 14 different clans representing different types of proteases with diverse active site residues.

  7. Photoelectrochemical CO2 reduction on 3C-SiC photoanode in aqueous solution

    NASA Astrophysics Data System (ADS)

    Song, Jun Tae; Iwasaki, Takayuki; Hatano, Mutsuko

    2015-04-01

    Photoelectrochemical (PEC) carbon dioxide (CO2) reduction on a 3C-SiC photoanode is demonstrated in aqueous solution with Pt and Ag counter electrodes. It is demonstrated that 3C-SiC has sufficient potential for CO2 reduction by confirming the band-edge structure. Then, the CO2 reduction is realized by connecting the 3C-SiC photoanode with the counter electrode. As the products of the PEC reaction with an applied bias of 1 V (vs counter electrode) to the 3C-SiC photoanode, hydrogen (H2) and carbon monoxide (CO) were analyzed by highly sensitive micro-gas chromatography, by which the time dependence of the gas products can be analyzed. Under light illumination of the 3C-SiC photoanode, CO2 reduction occurred while producing 2.5 and 9 nmol of CO gas with the Pt and Ag counter electrodes, respectively, after the reaction for 3000 s.

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

    NASA Astrophysics Data System (ADS)

    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) < 011 > BP||(100) < 011 > 3C-SiC and (111) < 11 2 ̅ > BP||(111) < 11 2 ̅ > 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.

  9. Unexpected Activity of a Novel Kunitz-type Inhibitor: INHIBITION OF CYSTEINE PROTEASES BUT NOT SERINE PROTEASES.

    PubMed

    Smith, David; Tikhonova, Irina G; Jewhurst, Heather L; Drysdale, Orla C; Dvořák, Jan; Robinson, Mark W; Cwiklinski, Krystyna; Dalton, John P

    2016-09-01

    Kunitz-type (KT) protease inhibitors are low molecular weight proteins classically defined as serine protease inhibitors. We identified a novel secreted KT inhibitor associated with the gut and parenchymal tissues of the infective juvenile stage of Fasciola hepatica, a helminth parasite of medical and veterinary importance. Unexpectedly, recombinant KT inhibitor (rFhKT1) exhibited no inhibitory activity toward serine proteases but was a potent inhibitor of the major secreted cathepsin L cysteine proteases of F. hepatica, FhCL1 and FhCL2, and of human cathepsins L and K (Ki = 0.4-27 nm). FhKT1 prevented the auto-catalytic activation of FhCL1 and FhCL2 and formed stable complexes with the mature enzymes. Pulldown experiments from adult parasite culture medium showed that rFhKT1 interacts specifically with native secreted FhCL1, FhCL2, and FhCL5. Substitution of the unusual P1 Leu(15) within the exposed reactive loop of FhKT1 for the more commonly found Arg (FhKT1Leu(15)/Arg(15)) had modest adverse effects on the cysteine protease inhibition but conferred potent activity against the serine protease trypsin (Ki = 1.5 nm). Computational docking and sequence analysis provided hypotheses for the exclusive binding of FhKT1 to cysteine proteases, the importance of the Leu(15) in anchoring the inhibitor into the S2 active site pocket, and the inhibitor's selectivity toward FhCL1, FhCL2, and human cathepsins L and K. FhKT1 represents a novel evolutionary adaptation of KT protease inhibitors by F. hepatica, with its prime purpose likely in the regulation of the major parasite-secreted proteases and/or cathepsin L-like proteases of its host.

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Highly potent fibrinolytic serine protease from Streptomyces.

    PubMed

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

    2011-01-01

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

  12. Schwann Cells Metabolize Extracellular 2',3'-cAMP to 2'-AMP.

    PubMed

    Verrier, Jonathan D; Kochanek, Patrick M; Jackson, Edwin K

    2015-08-01

    The 3',5'-cAMP-adenosine pathway (3',5'-cAMP→5'-AMP→adenosine) and the 2',3'-cAMP-adenosine pathway (2',3'-cAMP→2'-AMP/3'-AMP→adenosine) are active in the brain. Oligodendrocytes participate in the brain 2',3'-cAMP-adenosine pathway via their robust expression of 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNPase; converts 2',3'-cAMP to 2'-AMP). Because Schwann cells also express CNPase, it is conceivable that the 2',3'-cAMP-adenosine pathway exists in the peripheral nervous system. To test this and to compare the 2',3'-cAMP-adenosine pathway to the 3',5'-cAMP-adenosine pathway in Schwann cells, we examined the metabolism of 2',3'-cAMP, 2'-AMP, 3'-AMP, 3',5'-cAMP, and 5'-AMP in primary rat Schwann cells in culture. Addition of 2',3'-cAMP (3, 10, and 30 µM) to Schwann cells increased levels of 2'-AMP in the medium from 0.006 ± 0.002 to 21 ± 2, 70 ± 3, and 187 ± 10 nM/µg protein, respectively; in contrast, Schwann cells had little ability to convert 2',3'-cAMP to 3'-AMP or 3',5'-cAMP to either 3'-AMP or 5'-AMP. Although Schwann cells slightly converted 2',3'-cAMP and 2'-AMP to adenosine, they did so at very modest rates (e.g., 5- and 3-fold, respectively, more slowly compared with our previously reported studies in oligodendrocytes). Using transected myelinated rat sciatic nerves in culture medium, we observed a time-related increase in endogenous intracellular 2',3'-cAMP and extracellular 2'-AMP. These findings indicate that Schwann cells do not have a robust 3',5'-cAMP-adenosine pathway but do have a 2',3'-cAMP-adenosine pathway; however, because the pathway mostly involves 2'-AMP formation rather than 3'-AMP, and because the conversion of 2'-AMP to adenosine is slow, metabolism of 2',3'-cAMP mostly results in the accumulation of 2'-AMP. Accumulation of 2'-AMP in peripheral nerves postinjury could have pathophysiological consequences. PMID:25998049

  13. Design of translactam HCMV protease inhibitors as potent antivirals.

    PubMed

    Borthwick, Alan D

    2005-07-01

    Human cytomegalovirus (HCMV) is an important pathogen for which there is a significant unmet medical need. New HCMV antivirals, active against novel molecular targets, are undoubtedly needed as the currently available drugs ganciclovir, cidofovir, and foscarnet, which are all viral DNA inhibitors, suffer from limited effectiveness, mainly due to the development of drug resistance, poor bioavailability, and toxicity. One of the newer molecular targets that has been exploited in the search for better drug candidates is HCMV protease. Our deltaAla HCMV protease (wild type variant with the internal cleavage site deleted) was cloned and expressed in E. coli. This viral enzyme was used to develop HCMV protease assays to evaluate potential inhibitors. The chirally pure (SRS)-alpha-methyl pyrrolidine-5,5-trans-lactam template was synthesized, which together with the natural substrate requirements of HCMV protease and detailed SAR, was used to design potent and selective mechanism based inhibitors of HCMV protease. The mechanism of action of these inhibitors of HCMV protease was investigated by ESI/MS, and the X-ray crystal structure of the HCMV protease was used to refine our selective viral enzyme inhibitors to obtain plasma stable antivirals. A novel ELISA antiviral assay was developed which, together with a cytotoxicity assay, enabled us to discover anti-HCMV drug candidates equivalent in potency to ganciclovir that had good pharmacokinetics in the dog and good brain and ocular penetration in the guinea pig.

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

    PubMed

    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.

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

    PubMed

    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.

  16. Pioneer oral streptococci produce immunoglobulin A1 protease.

    PubMed Central

    Cole, M F; Evans, M; Fitzsimmons, S; Johnson, J; Pearce, C; Sheridan, M J; Wientzen, R; Bowden, G

    1994-01-01

    As part of a longitudinal study of the relationship between bacterial colonization and the secretory immune response, 367 isolates of pioneer viridans streptococci collected from 40 breast- and bottle-fed neonates within the first month postpartum were tested for the production of immunoglobulin A1 (IgA1) protease and glycosidases. Fifty percent of the streptococci isolated produced IgA1 protease, including all isolates of Streptococcus oralis and S. sanguis, 60.7% of S. mitis biovar 1 isolates, and some isolates that could not be identified. Three cleavage patterns of alpha 1 heavy chains were observed. Six isolates of S. mitis biovar 1 that did not produce IgA1 protease attacked the alpha 1 chain. Incubation of IgA1 protease-negative S. mitis biovar 1 isolates with IgA1, either prior to or together with S. sanguis, rendered the IgA1 paraprotein resistant to cleavage by the IgA1 protease of S. sanguis. The ability of some pioneer streptococci in the human oral cavity to produce IgA1 protease and of others to modify the susceptibility of IgA1 to cleavage by IgA1 protease perhaps enhances their ability to survive in this habitat. Images PMID:8188337

  17. Salt stress represses production of extracellular proteases in Bacillus pumilus.

    PubMed

    Liu, R F; Huang, C L; Feng, H

    2015-05-11

    Bacillus pumilus is able to secrete subtilisin-like prote-ases, one of which has been purified and characterized biochemically, demonstrating great potential for use in industrial applications. In the current study, the biosynthesis and transcription of extracellular pro-teases in B. pumilus (BA06) under salt stress were investigated using various methods, including a proteolytic assay, zymogram analysis, and real-time PCR. Our results showed that total extracellular proteolytic activity, both in fermentation broth and on milk-containing agar plates, was considerably repressed by salt in a dosage-dependent manner. As Bacillus species usually secret multiple extracellular proteases, a vari-ety of individual extracellular protease encoding genes were selected for real-time PCR analysis. It was shown that proteases encoded by the aprE and aprX genes were the major proteases in the fermentation broth in terms of their transcripts in B. pumilus. Further, transcription of aprE, aprX, and epr genes was indeed repressed by salt stress. In con-trast, transcription of other genes (e.g., vpr and wprA) was not repressed or significantly affected by the salt. Conclusively, salt stress represses total extracellular proteolytic activity in B. pumilus, which can largely be ascribed to suppression of the major protease-encoding genes (aprE, aprX) at the transcriptional level. In contrast, transcription of other pro-tease-encoding genes (e.g., vpr, wprA) was not repressed by salt stress.

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

    PubMed

    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

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    1999-09-01

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

  1. Staphylococcal proteases aid in evasion of the human complement system.

    PubMed

    Jusko, Monika; Potempa, Jan; Kantyka, Tomasz; Bielecka, Ewa; Miller, Halie K; Kalinska, Magdalena; Dubin, Grzegorz; Garred, Peter; Shaw, Lindsey N; Blom, Anna M

    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 in the hemolytic activity of serum, whereas two staphylococcal serine proteases D and E, had no effect. These four proteases were found to inhibit all pathways of complement due to the efficient degradation of several crucial components. Furthermore, S. aureus mutants lacking proteolytic enzymes were found to be more efficiently killed in human blood. Taken together, the major proteases of S. aureus appear to be important for pathogen-mediated evasion of the human complement system.

  2. Extracellular proteases modify cell wall turnover in Bacillus subtilis.

    PubMed Central

    Jolliffe, L K; Doyle, R J; Streips, U N

    1980-01-01

    The rate of turnover of peptidoglycan in exponentially growing cultures of Bacillus subtilis was observed to be sensitive to extracellular protease. In protease-deficient mutants the rates of cell wall turnover were greater than that of wild-type strain 168, whereas hyperprotease-producing strains exhibited decreased rates of peptidoglycan turnover. The rate of peptidogylcan turnover in a protease-deficient strain was decreased when the mutant was grown in the presence of a hyperprotease-producing strain. The addition of phenylmethylsulfonyl fluoride, a serine protease inhibitor, to cultures of hyperprotease-producing strains increased their rates of cell wall turnover. Isolated cell walls of all protease mutants contained autolysin levels equal to or greater than that of wild-type strain 168. The presence of filaments, or cells with incomplete septa, was observed in hyperprotease-producing strains or when a protease-deficient strain was grown in the presence of subtilisin. The results suggest that the turnover of cell walls in B. subtilis may be regulated by extracellular proteases. Images PMID:6102558

  3. Membrane Proteases and Aminoglycoside Antibiotic Resistance ▿ †

    PubMed Central

    Hinz, Aaron; Lee, Samuel; Jacoby, Kyle; Manoil, Colin

    2011-01-01

    We present genetic studies that help define the functional network underlying intrinsic aminoglycoside resistance in Pseudomonas aeruginosa. Our analysis shows that proteolysis, particularly that controlled by the membrane protease FtsH, is a major determinant of resistance. First, we examined the consequences of inactivating genes controlled by AmgRS, a two-component regulator required for intrinsic tobramycin resistance. Three of the gene products account for resistance: a modulator of FtsH protease (YccA), a membrane protease (HtpX), and a membrane protein of unknown function (PA5528). Second, we screened mutations inactivating 66 predicted proteases and related functions. Insertions inactivating two FtsH protease accessory factors (HflK and HflC) and a cytoplasmic protease (HslUV) increased tobramycin sensitivity. Finally, we generated an ftsH deletion mutation. The mutation dramatically increased aminoglycoside sensitivity. Many of the functions whose inactivation increased sensitivity appeared to act independently, since multiple mutations led to additive or synergistic effects. Up to 500-fold increases in tobramycin sensitivity were observed. Most of the mutations also were highly pleiotropic, increasing sensitivity to a membrane protein hybrid, several classes of antibiotics, alkaline pH, NaCl, and other compounds. We propose that the network of proteases provides robust protection from aminoglycosides and other substances through the elimination of membrane-disruptive mistranslation products. PMID:21764915

  4. Fluorometric CCHFV OTU protease assay with potent inhibitors.

    PubMed

    Kocabas, Fatih; Aslan, Galip S

    2015-10-01

    Crimean-Congo hemorrhagic fever virus (CCHFV) is a deadly virus that has been listed in the Category C as a potential bioterror agent. There are no specific therapies against CCHFV, which urges identification of potential therapeutic targets and development of CCHFV therapies. CCHFV OTU protease takes an important role in viral invasion through antagonizing NF-κB signaling. Inhibition of CCHFV OTU protease by small molecules warrants an exciting potential as antiviral therapeutics. Here we report the expression and purification of a C-His-tagged recombinant CCHFV OTU protease in E. coli BL21 (DE3) host strain. Activity of the refolded purified recombinant viral OTU protease has been validated with a UB-AMC fluorescent assay. In addition, we show a dose-dependent inhibition of the viral OTU protease by two small molecules. This study provides a reliable approach for recombinant expression and purification of CCHFV OTU protease, and demonstrates validation of OTU protease activity and its inhibition based on a UB-AMC florescent assay.

  5. Mengo virus 3C proteinase: recombinant expression, intergenus substrate cleavage and localization in vivo.

    PubMed

    Hall, D J; Palmenberg, A C

    1996-01-01

    Mengo virus 3C proteinase was cloned and expressed to high levels in a bacterial vector system. The protein was solubilized from inclusion bodies then purified to homogeneity (> 95%) by ion exchange chromatography. The recombinant enzyme was proteolytically active in cell-free processing assays with a Mengo capsid precursor substrate, L-P1-2A, correctly and proficiently cleaving it into L, 1AB, 1C, 1D and 2A protein products. Further analyses with synthetic peptide substrates encompassing the Mengo or rhinovirus-14 2C/3A cleavage sequences, showed the Mengo 3C could recognize and process specific glutamine-glycine sites within these peptides. The reactivity with the rhinovirus peptide was unexpected, because cross-reactivity between a picornavirus 3C enzyme and a protein substrate from different genus of this family has otherwise never been observed. In reciprocal reactions, a rhinovirus-14 3C preparation was unable to cleave the Mengo-derived synthetic peptide substrate. The recombinant Mengo 3C reactions were also characterized with regard to substrate Km, optimum pH and temperature. The protein was additionally used to raise monoclonal antibodies (mAbs) in mice, which in turn localized natural 3C, 3ABC, 3CD and P3 in immunoblots, immunoprecipitations and indirect immunofluorescence assays of Mengo-infected HeLa cells. The monoclonals showed cross-reactivity with 3C and 3C-containing precursors from encephalomyocarditis virus (EMCV), but did not react with 3C proteins from rhinovirus-14 or poliovirus-1M. PMID:8972564

  6. Protease inhibition as new therapeutic strategy for GI diseases

    PubMed Central

    Vergnolle, Nathalie

    2016-01-01

    The GI tract is the most exposed organ to proteases, both in physiological and pathophysiological conditions. For digestive purposes, the lumen of the upper GI tract contains large amounts of pancreatic proteases, but studies have also demonstrated increased proteolytic activity into mucosal tissues (both in the upper and lower GI tract), associated with pathological conditions. This review aims at outlining the evidences for dysregulated proteolytic homeostasis in GI diseases and the pathogenic mechanisms of increased proteolytic activity. The therapeutic potential of protease inhibition in GI diseases is discussed, with a particular focus on IBDs, functional GI disorders and colorectal cancer. PMID:27196587

  7. In vitro digestion with proteases producing MHC class II ligands.

    PubMed

    Tohmé, Mira; Maschalidi, Sophia; Manoury, Bénédicte

    2013-01-01

    Proteases generate peptides that bind to MHC class II molecules to interact with a wide diversity of CD4(+) T cells. They are expressed in dedicated organelles: endosomes and lysosomes of professional antigen presenting cells (pAPCs) such as B cells, macrophages, and dendritic cells. The identification of endosomal proteases which produce antigenic peptides is important, for example, for better vaccination and to prevent autoimmune diseases. Here, we describe a panel of technics (in vitro digestion assays of protein with recombinant proteases or purified endosomes/lysosomes, T cell stimulation) to monitor the production of MHC class II ligands. PMID:23329510

  8. Antiferromagnetic resonance in the Mott insulator fcc-Cs3C60.

    PubMed

    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.

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

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 3 2014-04-01 2014-04-01 false Mixed carbohydrase and protease enzyme product... Substances Affirmed as GRAS § 184.1027 Mixed carbohydrase and protease enzyme product. (a) Mixed carbohydrase and protease enzyme product is an enzyme preparation that includes carbohydrase and protease...

  10. Detection of Legume Protease Inhibitors by the Gel-X-ray Film Contact Print Technique

    ERIC Educational Resources Information Center

    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…

  11. Characterization of the protease activity of detergents: laboratory practicals for studying the protease profile and activity of various commercial detergents.

    PubMed

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

    2011-07-01

    Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body fluids, and food soils. This article describes two easy and cheap laboratory exercises to study the presence, profile, and basic enzymology of detergent proteases. These laboratory practicals are based on the determination of the detergent protease activity of various commercial detergents using the N-succinyl-L-alanyl-L-alanyl-L-prolyl-L-phenylalanine p-nitroanilide method and the bovine serum albumin degradation capacity. Students are also required to elucidate the enzymatic subtype of detergent proteases by studying the inhibitory potential of several types of protease inhibitors revealed by the same experimental methodology. Additionally, the results of the exercises can be used to provide additional insights on elementary enzymology by studying the influence of several important parameters on protease activity such as temperature (in this article) and the influence of pH and effects of surfactants and oxidizers (proposed). Students also develop laboratory skills, problem-solving capacities, and the ability to write a laboratory report. The exercises are mainly designed for an advanced undergraduate project in the biochemistry and biotechnology sciences. Globally, these laboratory practicals show students the biotechnological applications of proteases in the detergent industry and also reinforce important enzymology concepts.

  12. Heat resistant proteases produced in milk by psychrotrophic bacteria of dairy origin.

    PubMed

    Adams, D M; Barach, J T; Speck, M L

    1975-06-01

    Production of heat resistant proteases by psychrotrophs growing in milk, resistance of such proteases to ultrahigh temperature treatments and action of these enzymes on milk were studied. All of the psychrotrophs obtained from raw milk produced proteases that survived 149 C for 10s. Seventy to ninety percent of the raw milk samples contained psychrotrophs capable of producing heat resistant proteases. The protease chosen as a model was resistant to heat treatments at 110 to 150 C, and the inactivation parameters suggested that thermal destruction of heat resistant proteases would damage the milk severely. The casein content and pH of normal milk were suitable for protease action, and the protease was quite active at normal and elevated room temperatures. The protease rapidly spoiled sterile milk with the development of bitter flavor, clearing, or coagulation; and the susceptibility of sterile milk to protease increased during storage of the milk.

  13. Structural Basis for Molecular Discrimination by a 3',3'-cGAMP Sensing Riboswitch

    SciTech Connect

    Ren, Aiming; Wang, Xin  C.; Kellenberger, Colleen  A.; Rajashankar, Kanagalaghatta  R.; Jones, Roger  A.; Hammond, Ming  C.; Patel, Dinshaw  J.

    2015-04-07

    Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate the innate immune response in mammals. Besides protein receptors, there are bacterial riboswitches that selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch that targets 3',3'-cGAMP, which is distinguished from the endogenous mammalian signal 2',3'-cGAMP by its backbone connectivity. Here, we report on structures of the aptamer domain of the 3',3'-cGAMP riboswitch from Geobacter in the 3',3'-cGAMP and c-di-GMP bound states. The riboswitch adopts a tuning forklike architecture with a junctional ligand-binding pocket and different orientations of the arms are correlated with the identity of the bound cyclic dinucleotide. Subsequent biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, which has implications for both the assignment and reengineering of riboswitches in this structural class.

  14. Are 3C 120 and Other Active Galactic Nuclei Overweight Microquasars?

    NASA Astrophysics Data System (ADS)

    Marscher, Alan P.

    2005-11-01

    The appearance of superluminal radio knots follows drops in the X-ray flux in the FR1 radio galaxy 3C 120 and possibly the FR2 source 3C 111. This corresponds in a very general way to the behavior of the X-ray binary GRS 1915 + 105, but the light curves of the microquasar are much richer in detail. Starting in 2003.7, the character of the radio and X-ray light curves of 3C 120 changed, perhaps signaling a new stage of activity. I discuss here what one might expect when a microquasar is scaled up to AGN dimensions, and compare this with what we see in 3C 120. There is a mismatch between expectations and observations.

  15. Expression and secretion of heterologous proteases by Corynebacterium glutamicum.

    PubMed Central

    Billman-Jacobe, H; Wang, L; Kortt, A; Stewart, D; Radford, A

    1995-01-01

    Genes encoding the basic protease of Dichelobacter nodosus (bprV) and the subtilisin of Bacillus subtilis (aprE) were cloned and expressed in Corynebacterium glutamicum. In each case, enzymatically active protein was detected in the supernatants of liquid cultures. While the secretion of subtilisin was directed by its own signal peptide, the natural signal peptide of the bprV basic protease did not facilitate secretion. A hybrid aprE-bprV gene in which the promoter and signal peptide coding sequences of subtilisin replaced those of bprV could be expressed, and basic protease was secreted by C. glutamicum. Expression of these proteases in C. glutamicum provides an opportunity to compare protein secretion from this gram-positive host with that from other gram-positive and gram-negative bacteria. PMID:7747974

  16. Genotype dependent QSAR for HIV-1 protease inhibition.

    PubMed

    Boutton, Carlo W; De Bondt, Hendrik L; De Jonge, Marc R

    2005-03-24

    The development of drug-resistant viruses limits the therapeutic success of anti-HIV therapies. Some of these genetic HIV-variants display complex mutational patterns in their pol gene that codes for protease and reverse transcriptase, the most investigated molecular targets for antiretroviral therapy. In this paper, we present a computational structure-based approach to predict the resistance of a HIV-1 protease strain to amprenavir by calculating the interaction energy of the drug with HIV-1 protease. By considering the interaction energy per residue, we can identify what residue mutations contribute to drug-resistance. This approach is presented here as a structure-based tool for the prediction of resistance of HIV-1 protease toward amprenavir, with a view to use the drug-protein interaction-energy pattern in a lead-optimization procedure for the discovery of new anti-HIV drugs. PMID:15771454

  17. Improving Viral Protease Inhibitors to Counter Drug Resistance.

    PubMed

    Kurt Yilmaz, Nese; Swanstrom, Ronald; Schiffer, Celia A

    2016-07-01

    Drug resistance is a major problem in health care, undermining therapy outcomes and necessitating novel approaches to drug design. Extensive studies on resistance to viral protease inhibitors, particularly those of HIV-1 and hepatitis C virus (HCV) protease, revealed a plethora of information on the structural and molecular mechanisms underlying resistance. These insights led to several strategies to improve viral protease inhibitors to counter resistance, such as exploiting the essential biological function and leveraging evolutionary constraints. Incorporation of these strategies into structure-based drug design can minimize vulnerability to resistance, not only for viral proteases but for other quickly evolving drug targets as well, toward designing inhibitors one step ahead of evolution to counter resistance with more intelligent and rational design. PMID:27090931

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

    PubMed

    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.

  19. Target Cell APOBEC3C Can Induce Limited G-to-A Mutation in HIV-1

    PubMed Central

    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

  20. AN ASYMMETRICAL SYNCHROTRON MODEL FOR KNOTS IN THE 3C 273 JET

    SciTech Connect

    Liu, Wen-Po; Chen, Y. J.; Wang, Chun-Cheng

    2015-06-20

    To interpret the emission of knots in the 3C 273 jet from radio to X-rays, we propose a synchrotron model in which, owing to the shock compression effect, the injection spectra from a shock into the upstream and downstream emission regions are asymmetric. Our model could well explain the spectral energy distributions of knots in the 3C 273 jet, and predictions regarding the knots’ spectra could be tested by future observations.

  1. Picornaviral 3C cysteine proteinases have a fold similar to the chymotrypsin-like serine proteinases

    SciTech Connect

    Allaire,M.; Chernaia, M.; Malcolm, B.; James, M.

    1994-01-01

    The picornavirus family includes several pathogens such as poliovirus, rhinovirus (the major cause of the common cold), hepatitis A virus and the foot-and-mouth disease virus. Picornaviral proteins are expressed by direct translation of the genomic RNA into a single, large polyprotein precursor. Proteolysis of the viral polyprotein into the mature proteins is assured by the viral 3C enzymes, which are cysteine proteinases. Here we report the X-ray crystal structure at 2.3 {angstrom} resolution of the 3C proteinase from hepatitis A virus (HAV-3C). The overall architecture of HAV-3C reveals a fold resembling that of the chymotrypsin family of serine proteinases, which is consistent with earlier predictions. Catalytic residues include Cys 172 as nucleophile and His 44 as general base. The 3C cleavage specificity for glutamine residues is defined primarily by His 191. The overall structure suggests that an inter-molecular (trans) cleavage releases 3C and that there is an active proteinase in the polyprotein.

  2. Diabetes mellitus secondary to pancreatic diseases (Type 3c)--are we neglecting an important disease?

    PubMed

    Ewald, Nils; Bretzel, Reinhard G

    2013-04-01

    Type 3c diabetes mellitus (T3cDM) is a clinically relevant condition with a prevalence of 5-10% among all diabetic subjects in Western populations. Its prevalence and clinical importance have been underestimated and underappreciated so far. In contrast to the management of type 1 or type 2 diabetes, the endocrinopathy in T3cDM is very complex and complicated by additional present comorbidities such as maldigestion and concommitant qualitative malnutrition. The failure to correctly diagnose T3cDM leads to failure to implement an appropriate medical therapy of these patients. Physicians should screen for important and easily reversable pathological conditions such as exocrine insufficiency, lack of fat-soluble vitamins (especially vitamin D) and impairment of fat hydrolysis and incretin secretion which are found very commonly in T3cDM. Since most patients with T3cDM suffer from chronic pancreatitis, physicians must additionally be aware of the elevated risk of pancreatic cancer in this subset of patients.

  3. Cleavage of interferon regulatory factor 7 by enterovirus 71 3C suppresses cellular responses.

    PubMed

    Lei, Xiaobo; Xiao, Xia; Xue, Qinghua; Jin, Qi; He, Bin; Wang, Jianwei

    2013-02-01

    Enterovirus 71 (EV71) is a positive-stranded RNA virus which is capable of inhibiting innate immunity. Among virus-encoded proteins, the 3C protein compromises the type I interferon (IFN-I) response mediated by retinoid acid-inducible gene-I (RIG-I) or Toll-like receptor 3 that activates interferon regulatory 3 (IRF3) and IRF7. In the present study, we report that enterovirus 71 downregulates IRF7 through the 3C protein, which inhibits the function of IRF7. When expressed in mammalian cells, the 3C protein mediates cleavage of IRF7 rather than that of IRF3. This process is insensitive to inhibitors of caspase, proteasome, lysosome, and autophagy. H40D substitution in the 3C active site abolishes its activity, whereas R84Q or V154S substitution in the RNA binding motif has no effect. Furthermore, 3C-mediated cleavage occurs at the Q189-S190 junction within the constitutive activation domain of IRF7, resulting in two cleaved IRF7 fragments that are incapable of activating IFN expression. Ectopic expression of wild-type IRF7 limits EV71 replication. On the other hand, expression of the amino-terminal domain of IRF7 enhances EV71 infection, which correlates with its ability to interact with and inhibit IRF3. These results suggest that control of IRF7 by the 3C protein may represent a viral mechanism to escape cellular responses. PMID:23175366

  4. Synthesis and electrochemical performance of Ti3C2Tx with hydrothermal process

    NASA Astrophysics Data System (ADS)

    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.

  5. Binary WC- and Cr3C2-containing hardmetal compositions for thermally sprayed coatings

    NASA Astrophysics Data System (ADS)

    Berger, L.-M.

    2016-03-01

    Compositions of thermally sprayed hardmetal coatings for wear protection are based on the hard materials WC and Cr3C2 with Co and Ni as the most important binders, which are often alloyed with Cr. There are a few commercial compositions containing WC and Cr3C2 together, which have a high potential for the improvement of coating properties, in particular for service in corrosive environments and high temperature applications. However, the combined application of WC and Cr3C2 in the coating compositions leads to very complex reactions between these components both during feedstock powder preparation and the spray process. This contribution summarizes the knowledge about the interactions of WC and Cr3C2 for the most important commercially available compositions: WC-10Co-4Cr, WC-20‘CrC’-7Ni, 45Cr3C2- 37WC-18NiCo. These three compositions show remarkable differences of the interaction between WC and Cr3C2 and the coating properties.

  6. Synthesis and electrochemical performance of Ti3C2Tx with hydrothermal process

    NASA Astrophysics Data System (ADS)

    Wang, Libo; Zhang, Heng; Wang, Bo; Shen, Changjie; Zhang, Chuanxiang; Hu, Qianku; Zhou, Aiguo; Liu, Baozhong

    2016-08-01

    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. [Figure not available: see fulltext.

  7. Atomic-scale recognition of surface structure and intercalation mechanism of Ti3C2X.

    PubMed

    Wang, Xuefeng; Shen, Xi; Gao, Yurui; Wang, Zhaoxiang; Yu, Richeng; Chen, Liquan

    2015-02-25

    MXenes represent a large family of functionalized two-dimensional (2D) transition-metal carbides and carbonitrides. However, most of the understanding on their unique structures and applications stops at the theoretical suggestion and lack of experimental support. Herein, the surface structure and intercalation chemistry of Ti3C2X are clarified at the atomic scale by aberration-corrected scanning transmission electron microscope (STEM) and density functional theory (DFT) calculations. The STEM studies show that the functional groups (e.g., OH(-), F(-), O(-)) and the intercalated sodium (Na) ions prefer to stay on the top sites of the centro-Ti atoms and the C atoms of the Ti3C2 monolayer, respectively. Double Na-atomic layers are found within the Ti3C2X interlayer upon extensive Na intercalation via two-phase transition and solid-solution reactions. In addition, aluminum (Al)-ion intercalation leads to horizontal sliding of the Ti3C2X monolayer. On the basis of these observations, the previous monolayer surface model of Ti3C2X is modified. DFT calculations using the new modeling help to understand more about their physical and chemical properties. These findings enrich the understanding of the MXenes and shed light on future material design and applications. Moreover, the Ti3C2X exhibits prominent rate performance and long-term cycling stability as an anode material for Na-ion batteries. PMID:25688582

  8. Characterization of the immunoglobulin A protease of Ureaplasma urealyticum.

    PubMed Central

    Spooner, R K; Russell, W C; Thirkell, D

    1992-01-01

    Ureaplasma urealyticum strains of all serotypes express a specific human immunoglobulin A1 protease that cleaves immunoglobulin A1 to produce intact Fab and Fc fragments. The use of a variety of inhibitors suggests that the enzyme is a serine protease. N-terminal sequencing of the Fc digestion product showed that the enzyme cleaves between the proline and threonine residues 235 and 236 in the hinge region of the heavy chain of immunoglobulin A1. Images PMID:1587621

  9. Proteomic Substrate Identification for Membrane Proteases in the Brain

    PubMed Central

    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. PMID:27790089

  10. Amplified detection of protease activity using porous silicon nanostructures

    NASA Astrophysics Data System (ADS)

    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.

  11. 2-D zymographic analysis of Broccoli (Brassica oleracea L. var. Italica) florets proteases: follow up of cysteine protease isotypes in the course of post-harvest senescence.

    PubMed

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

    2011-09-01

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

  12. Dual origin of gut proteases in Formosan subterranean termites (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae).

    PubMed

    Sethi, Amit; Xue, Qing-Gang; La Peyre, Jerome F; Delatte, Jennifer; Husseneder, Claudia

    2011-07-01

    Cellulose digestion in lower termites, mediated by carbohydrases originating from both termite and endosymbionts, is well characterized. In contrast, limited information exists on gut proteases of lower termites, their origins and roles in termite nutrition. The objective of this study was to characterize gut proteases of the Formosan subterranean termite (Coptotermes formosanus Shiraki) (Isoptera: Rhinotermitidae). The protease activity of extracts from gut tissues (fore-, mid- and hindgut) and protozoa isolated from hindguts of termite workers was quantified using hide powder azure as a substrate and further characterized by zymography with gelatin SDS-PAGE. Midgut extracts showed the highest protease activity followed by the protozoa extracts. High level of protease activity was also detected in protozoa culture supernatants after 24 h incubation. Incubation of gut and protozoa extracts with class-specific protease inhibitors revealed that most of the proteases were serine proteases. All proteolytic bands identified after gelatin SDS-PAGE were also inhibited by serine protease inhibitors. Finally, incubation with chromogenic substrates indicated that extracts from fore- and hindgut tissues possessed proteases with almost exclusively trypsin-like activity while both midgut and protozoa extracts possessed proteases with trypsin-like and subtilisin/chymotrypsin-like activities. However, protozoa proteases were distinct from midgut proteases (with different molecular mass). Our results suggest that the Formosan subterranean termite not only produces endogenous proteases in its gut tissues, but also possesses proteases originating from its protozoan symbionts.

  13. Proteases of germinating winged-bean (Psophocarpus tetragonolobus) seeds: purification and characterization of an acidic protease.

    PubMed

    Usha, R; Singh, M

    1996-01-15

    Two major classes of protease are shown to occur in germinating winged-bean (Psophocarpus tetragonolobus) seeds, by assaying extracts at pH 8.0 and pH 5.1 with [14C]gelatin as substrate. At pH 8.0, the activity profile of the enzyme shows a steady rise throughout the period of germination, whereas the activity at the acidic pH is very low up to day 5 and then increases sharply reaching a peak on day 11, followed by an equally sharp decline. The winged-bean acidic protease (WbAP) has been purified to apparent homogeneity, as attested by a single protein band on both PAGE and SDS/PAGE. WbAP is a monomeric enzyme with a molecular mass of 35 kDa and a pH optimum of 6.0. It is a thiol protease that does not belong to the papain family and it has tightly bound Ca2+ as shown by 45Ca(2+)-exchange studies. Besides gelatin and casein, it hydrolyses a 29 kDa winged-bean protein, indicating a prospective physiological role for it in storage-protein mobilization. Immunoblot analysis shows that it occurs only in the seeds and sprouting tubers of this plant and also that it is synthesized in developing seeds just before desiccation. It appears that the newly synthesized enzyme is inactive, and activation takes place around day 6 of germination. However, neither the mechanism of activation nor the signal that triggers it is clearly understood.

  14. The crystal structure of GXGD membrane protease FlaK

    SciTech Connect

    Hu, Jian; Xue, Yi; Lee, Sangwon; Ha, Ya

    2011-09-20

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  15. The Crystal Structure of GXGD Membrane Protease FlaK

    SciTech Connect

    J Hu; Y Xue; S Lee; Y Ha

    2011-12-31

    The GXGD proteases are polytopic membrane proteins with catalytic activities against membrane-spanning substrates that require a pair of aspartyl residues. Representative members of the family include preflagellin peptidase, type 4 prepilin peptidase, presenilin and signal peptide peptidase. Many GXGD proteases are important in medicine. For example, type 4 prepilin peptidase may contribute to bacterial pathogenesis, and mutations in presenilin are associated with Alzheimer's disease. As yet, there is no atomic-resolution structure in this protease family. Here we report the crystal structure of FlaK, a preflagellin peptidase from Methanococcus maripaludis, solved at 3.6 {angstrom} resolution. The structure contains six transmembrane helices. The GXGD motif and a short transmembrane helix, helix 4, are positioned at the centre, surrounded by other transmembrane helices. The crystal structure indicates that the protease must undergo conformational changes to bring the GXGD motif and a second essential aspartyl residue from transmembrane helix 1 into close proximity for catalysis. A comparison of the crystal structure with models of presenilin derived from biochemical analysis reveals three common transmembrane segments that are similarly arranged around the active site. This observation reinforces the idea that the prokaryotic and human proteases are evolutionarily related. The crystal structure presented here provides a framework for understanding the mechanism of the GXGD proteases, and may facilitate the rational design of inhibitors that target specific members of the family.

  16. The roles of intramembrane proteases in protozoan parasites.

    PubMed

    Sibley, L David

    2013-12-01

    Intramembrane proteolysis is widely conserved throughout different forms of life, with three major types of proteases being known for their ability to cleave peptide bonds directly within the transmembrane domains of their substrates. Although intramembrane proteases have been extensively studied in humans and model organisms, they have only more recently been investigated in protozoan parasites, where they turn out to play important and sometimes unexpected roles. Signal peptide peptidases are involved in endoplasmic reticulum (ER) quality control and signal peptide degradation from exported proteins. Recent studies suggest that repurposing inhibitors developed for blocking presenilins may be useful for inhibiting the growth of Plasmodium, and possibly other protozoan parasites, by blocking signal peptide peptidases. Rhomboid proteases, originally described in the fly, are also widespread in parasites, and are especially expanded in apicomplexans. Their study in parasites has revealed novel roles that expand our understanding of how these proteases function. Within this diverse group of parasites, rhomboid proteases contribute to processing of adhesins involved in attachment, invasion, intracellular replication, phagocytosis, and immune evasion, placing them at the vertex of host-parasite interactions. This article is part of a Special Issue entitled: Intramembrane Proteases.

  17. Insights into the Cyanobacterial Deg/HtrA Proteases

    PubMed Central

    Cheregi, Otilia; Wagner, Raik; Funk, Christiane

    2016-01-01

    Proteins are the main machinery for all living processes in a cell; they provide structural elements, regulate biochemical reactions as enzymes, and are the interface to the outside as receptors and transporters. Like any other machinery proteins have to be assembled correctly and need maintenance after damage, e.g., caused by changes in environmental conditions, genetic mutations, and limitations in the availability of cofactors. Proteases and chaperones help in repair, assembly, and folding of damaged and misfolded protein complexes cost-effective, with low energy investment compared with neo-synthesis. Despite their importance for viability, the specific biological role of most proteases in vivo is largely unknown. Deg/HtrA proteases, a family of serine-type ATP-independent proteases, have been shown in higher plants to be involved in the degradation of the Photosystem II reaction center protein D1. The objective of this review is to highlight the structure and function of their cyanobacterial orthologs. Homology modeling was used to find specific features of the SynDeg/HtrA proteases of Synechocystis sp. PCC 6803. Based on the available data concerning their location and their physiological substrates we conclude that these Deg proteases not only have important housekeeping and chaperone functions within the cell, but also are needed for remodeling the cell exterior. PMID:27252714

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

    PubMed

    Schauperl, Michael; Fuchs, Julian E; Waldner, Birgit J; Huber, Roland G; Kramer, Christian; Liedl, Klaus R

    2015-01-01

    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. PMID:26559682

  19. Signaling pathways activated by a protease allergen in basophils

    PubMed Central

    Rosenstein, Rachel K.; Bezbradica, Jelena S.; Yu, Shuang; Medzhitov, Ruslan

    2014-01-01

    Allergic diseases represent a significant burden in industrialized countries, but why and how the immune system responds to allergens remain largely unknown. Because many clinically significant allergens have proteolytic activity, and many helminths express proteases that are necessary for their life cycles, host mechanisms likely have evolved to detect the proteolytic activity of helminth proteases, which may be incidentally activated by protease allergens. A cysteine protease, papain, is a prototypic protease allergen that can directly activate basophils and mast cells, leading to the production of cytokines, including IL-4, characteristic of the type 2 immune response. The mechanism of papain’s immunogenic activity remains unknown. Here we have characterized the cellular response activated by papain in basophils. We find that papain-induced IL-4 production requires calcium flux and activation of PI3K and nuclear factor of activated T cells. Interestingly, papain-induced IL-4 production was dependent on the immunoreceptor tyrosine-based activation motif (ITAM) adaptor protein Fc receptor γ-chain, even though the canonical ITAM signaling was not activated by papain. Collectively, these data characterize the downstream signaling pathway activated by a protease allergen in basophils. PMID:25369937

  20. Image reconstruction algorithm for optically stimulated luminescence 2D dosimetry using laser-scanned Al2O3:C and Al2O3:C,Mg films

    NASA Astrophysics Data System (ADS)

    Ahmed, M. F.; Schnell, E.; Ahmad, S.; Yukihara, E. G.

    2016-10-01

    The objective of this work was to develop an image reconstruction algorithm for 2D dosimetry using Al2O3:C and Al2O3:C,Mg optically stimulated luminescence (OSL) films imaged using a laser scanning system. The algorithm takes into account parameters associated with detector properties and the readout system. Pieces of Al2O3:C films (~8 mm  ×  8 mm  ×  125 µm) were irradiated and used to simulate dose distributions with extreme dose gradients (zero and non-zero dose regions). The OSLD film pieces were scanned using a custom-built laser-scanning OSL reader and the data obtained were used to develop and demonstrate a dose reconstruction algorithm. The algorithm includes corrections for: (a) galvo hysteresis, (b) photomultiplier tube (PMT) linearity, (c) phosphorescence, (d) ‘pixel bleeding’ caused by the 35 ms luminescence lifetime of F-centers in Al2O3, (e) geometrical distortion inherent to Galvo scanning system, and (f) position dependence of the light collection efficiency. The algorithm was also applied to 6.0 cm  ×  6.0 cm  ×  125 μm or 10.0 cm  ×  10.0 cm  ×  125 µm Al2O3:C and Al2O3:C,Mg films exposed to megavoltage x-rays (6 MV) and 12C beams (430 MeV u‑1). The results obtained using pieces of irradiated films show the ability of the image reconstruction algorithm to correct for pixel bleeding even in the presence of extremely sharp dose gradients. Corrections for geometric distortion and position dependence of light collection efficiency were shown to minimize characteristic limitations of this system design. We also exemplify the application of the algorithm to more clinically relevant 6 MV x-ray beam and a 12C pencil beam, demonstrating the potential for small field dosimetry. The image reconstruction algorithm described here provides the foundation for laser-scanned OSL applied to 2D dosimetry.

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

    PubMed Central

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

    2016-01-01

    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. PMID:26955973

  2. Role of CNPase in the oligodendrocytic extracellular 2',3'-cAMP-adenosine pathway.

    PubMed

    Verrier, Jonathan D; Jackson, Travis C; Gillespie, Delbert G; Janesko-Feldman, Keri; Bansal, Rashmi; Goebbels, Sandra; Nave, Klaus-Armin; Kochanek, Patrick M; Jackson, Edwin K

    2013-10-01

    Extracellular adenosine 3',5'-cyclic monophosphate (3',5'-cAMP) is an endogenous source of localized adenosine production in many organs. Recent studies suggest that extracellular 2',3'-cAMP (positional isomer of 3',5'-cAMP) is also a source of adenosine, particularly in the brain in vivo post-injury. Moreover, in vitro studies show that both microglia and astrocytes can convert extracellular 2',3'-cAMP to adenosine. Here, we examined the ability of primary mouse oligodendrocytes and neurons to metabolize extracellular 2',3'-cAMP and their respective adenosine monophosphates (2'-AMP and 3'-AMP). Cells were also isolated from mice deficient in 2',3'-cyclic nucleotide-3'-phosphodiesterase (CNPase). Oligodendrocytes metabolized 2',3'-cAMP to 2'-AMP with 10-fold greater efficiency than did neurons (and also more than previously examined microglia and astrocytes); whereas, the production of 3'-AMP was minimal in both oligodendrocytes and neurons. The production of 2'-AMP from 2',3'-cAMP was reduced by 65% in CNPase -/- versus CNPase +/+ oligodendrocytes. Oligodendrocytes also converted 2'-AMP to adenosine, and this was also attenuated in CNPase -/- oligodendrocytes. Inhibition of classic 3',5'-cAMP-3'-phosphodiesterases with 3-isobutyl-1-methylxanthine did not block metabolism of 2',3'-cAMP to 2'-AMP and inhibition of classic ecto-5'-nucleotidase (CD73) with α,β-methylene-adenosine-5'-diphosphate did not attenuate the conversion of 2'-AMP to adenosine. These studies demonstrate that oligodendrocytes express the extracellular 2',3'-cAMP-adenosine pathway (2',3'-cAMP → 2'-AMP → adenosine). This pathway is more robustly expressed in oligodendrocytes than in all other CNS cell types because CNPase is the predominant enzyme that metabolizes 2',3'-cAMP to 2-AMP in CNS cells. By reducing levels of 2',3'-cAMP (a mitochondrial toxin) and increasing levels of adenosine (a neuroprotectant), oligodendrocytes may protect axons from injury. PMID:23922219

  3. Protease Production by Different Thermophilic Fungi

    NASA Astrophysics Data System (ADS)

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

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

  4. Cystatin protease inhibitors and immune functions.

    PubMed

    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.

  5. Pyrazinone protease inhibitor metabolites from Photorhabdus luminescens.

    PubMed

    Park, Hyun Bong; Crawford, Jason M

    2016-08-01

    Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that undergoes phenotypic variation and lives in mutualistic association with nematodes of the family Heterorhabditidae. The pair infects and kills insects, and during their coordinated lifecycle, the bacteria produce an assortment of specialized metabolites to regulate its mutualistic and pathogenic roles. As part of our search for new specialized metabolites from the Photorhabdus genus, we examined organic extracts from P. luminescens grown in an amino-acid-rich medium based on the free amino-acid levels found in the circulatory fluid of its common insect prey, the Galleria mellonella larva. Reversed-phase HPLC/UV/MS-guided fractionation of the culture extracts led to the identification of two new pyrazinone metabolites, lumizinones A (1) and B (2), together with two N-acetyl dipeptides (3 and 4). The lumizinones were produced only in the phenotypic variant associated with nematode development and insect pathogenesis. Their chemical structures were elucidated by analysis of 1D and 2D NMR and high-resolution ESI-QTOF-MS spectral data. The absolute configurations of the amino acids in 3 and 4 were determined by Marfey's analysis. Compounds 1-4 were evaluated for their calpain protease inhibitory activity, and lumizinone A (1) showed inhibition with an IC50 (half-maximal inhibitory concentration) value of 3.9 μm. PMID:27353165

  6. Fragment-Based Screen against HIV Protease

    PubMed Central

    Perryman, A. L.; Zhang, Q.; Soutter, H. H.; Rosenfeld, R.; McRee, D. E.; Olson, A. J.; Elder, J. E.; Stout, C. D.

    2009-01-01

    We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 Å resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the ‘exo site’ adjacent to the Gly16Gly17Gln18 loop where the amide of Gly17 is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys14 and Leu63. Another fragment, indole-6-carboxylic acid, binds on the ‘outside/top of the flap’ via hydrophobic contacts with Trp42, Pro44, Met46, and Lys55, a hydrogen bond with Val56, and a salt-bridge with Arg57. 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target. PMID:20659109

  7. Fragment-based screen against HIV protease.

    PubMed

    Perryman, Alexander L; Zhang, Qing; Soutter, Holly H; Rosenfeld, Robin; McRee, Duncan E; Olson, Arthur J; Elder, John E; Stout, C David

    2010-03-01

    We have employed a fragment-based screen against wild-type (NL4-3) HIV protease (PR) using the Active Sight fragment library and X-ray crystallography. The experiments reveal two new binding sites for small molecules. PR was co-crystallized with fragments, or crystals were soaked in fragment solutions, using five crystal forms, and 378 data sets were collected to 2.3-1.3 A resolution. Fragment binding induces a distinct conformation and specific crystal form of TL-3 inhibited PR during co-crystallization. One fragment, 2-methylcyclohexanol, binds in the 'exo site' adjacent to the Gly(16)Gly(17)Gln(18)loop where the amide of Gly(17)is a specific hydrogen bond donor, and hydrophobic contacts occur with the side chains of Lys(14)and Leu(63). Another fragment, indole-6-carboxylic acid, binds on the 'outside/top of the flap' via hydrophobic contacts with Trp(42), Pro(44), Met(46), and Lys(55), a hydrogen bond with Val(56), and a salt-bridge with Arg(57). 2-acetyl-benzothiophene also binds at this site. This study is the first fragment-based crystallographic screen against HIV PR, and the first time that fragments were screened against an inhibitor-bound drug target to search for compounds that both bind to novel sites and stabilize the inhibited conformation of the target.

  8. Antifungal effects of synthetic human β-defensin 3-C15 peptide

    PubMed Central

    Ahn, Ki-Bum; Kim, Christine; Kum, Jong-Won; Gu, Yu; Han, Seung Hyun; Shon, Won-Jun; Lee, Woocheol; Zhu, Qiang

    2016-01-01

    Objectives The purpose of this ex vivo study was to compare the antifungal activity of a synthetic peptide consisting of 15 amino acids at the C-terminus of human β-defensin 3 (HBD3-C15) with calcium hydroxide (CH) and Nystatin (Nys) against Candida albicans (C. albicans) biofilm. Materials and Methods C. albicans were grown on cover glass bottom dishes or human dentin disks for 48 hr, and then treated with HBD3-C15 (0, 12.5, 25, 50, 100, 150, 200, and 300 µg/mL), CH (100 µg/mL), and Nys (20 µg/mL) for 7 days at 37℃. On cover glass, live and dead cells in the biomass were measured by the FilmTracer Biofilm viability assay, and observed by confocal laser scanning microscopy (CLSM). On dentin, normal, diminished and ruptured cells were observed by field-emission scanning electron microscopy (FE-SEM). The results were subjected to a two-tailed t-test, a one way analysis variance and a post hoc test at a significance level of p = 0.05. Results C. albicans survival on dentin was inhibited by HBD3-C15 in a dose-dependent manner. There were fewer aggregations of C. albicans in the groups of Nys and HBD3-C15 (≥ 100 µg/mL). CLSM showed C. albicans survival was reduced by HBD3-C15 in a dose dependent manner. Nys and HBD3-C15 (≥ 100 µg/mL) showed significant fungicidal activity compared to CH group (p < 0.05). Conclusions Synthetic HBD3-C15 peptide (≥ 100 µg/mL) and Nys exhibited significantly higher antifungal activity than CH against C. albicans by inhibiting cell survival and biofilm. PMID:27200276

  9. Experimental Investigation of the Cr3C2 C Peritectic Fixed Point

    NASA Astrophysics Data System (ADS)

    Zheng, W.; Yamada, Y.; Wang, Y.

    2008-06-01

    The Cr3C2 C (1,826°C) peritectic point was investigated for its performance as a high-temperature fixed point. Dependence on the impurity content was observed, although it was less severe for the higher of the two equilibrium temperatures obtained with the same cell, the Cr3C2 C peritectic point, than for the lower, the Cr7C3 Cr3C2 eutectic point. Thermal history had an effect on the melting plateau duration, but not on the point-of-inflection temperature nor on the melting range. The melting rate had no apparent effect. The repeatability evaluated as the standard deviation of the repeated melting plateaux within a day was 20 mK for the Cr3C2 C peritectic point, while for the Cr7C3 Cr3C2 eutectic point, this was 210 mK. For both the Cr3C2 C peritectic and the Cr7C3 Cr3C2 eutectic, the freezing plateaux often showed deep supercools, which made them unsuitable for use. The observed good repeatability shows the peritectic-point performance to be comparable to the best MC-eutectic high-temperature fixed points investigated so far. The insensitivity to thermal history constitutes an important and practical advantage. The low price of chromium is a clear benefit as compared to Pt C (1,738°C) or Ru C (1,953°C) eutectic points, the M C eutectic points in this temperature range.

  10. Protease induced plasticity: matrix metalloproteinase-1 promotes neurostructural changes through activation of protease activated receptor 1

    PubMed Central

    Allen, Megan; Ghosh, Suhasini; Ahern, Gerard P.; Villapol, Sonia; Maguire-Zeiss, Kathleen A.; Conant, Katherine

    2016-01-01

    Matrix metalloproteinases (MMPs) are a family of secreted endopeptidases expressed by neurons and glia. Regulated MMP activity contributes to physiological synaptic plasticity, while dysregulated activity can stimulate injury. Disentangling the role individual MMPs play in synaptic plasticity is difficult due to overlapping structure and function as well as cell-type specific expression. Here, we develop a novel system to investigate the selective overexpression of a single MMP driven by GFAP expressing cells in vivo. We show that MMP-1 induces cellular and behavioral phenotypes consistent with enhanced signaling through the G-protein coupled protease activated receptor 1 (PAR1). Application of exogenous MMP-1, in vitro, stimulates PAR1 dependent increases in intracellular Ca2+ concentration and dendritic arborization. Overexpression of MMP-1, in vivo, increases dendritic complexity and induces biochemical and behavioral endpoints consistent with increased GPCR signaling. These data are exciting because we demonstrate that an astrocyte-derived protease can influence neuronal plasticity through an extracellular matrix independent mechanism. PMID:27762280

  11. A Single Nucleotide Polymorphism in Human APOBEC3C Enhances Restriction of Lentiviruses

    PubMed Central

    Wittkopp, Cristina J.; Adolph, Madison B.; Wu, Lily I.; Chelico, Linda; Emerman, Michael

    2016-01-01

    Humans express seven human APOBEC3 proteins, which can inhibit viruses and endogenous retroelements through cytidine deaminase activity. The seven paralogs differ in the potency of their antiviral effects, as well as in their antiviral targets. One APOBEC3, APOBEC3C, is exceptional as it has been found to only weakly block viruses and endogenous retroelements compared to other APOBEC3s. However, our positive selection analyses suggest that APOBEC3C has played a role in pathogen defense during primate evolution. Here, we describe a single nucleotide polymorphism in human APOBEC3C, a change from serine to isoleucine at position 188 (I188) that confers potent antiviral activity against HIV-1. The gain-of-function APOBEC3C SNP results in increased enzymatic activity and hypermutation of target sequences when tested in vitro, and correlates with increased dimerization of the protein. The I188 is widely distributed in human African populations, and is the ancestral primate allele, but is not found in chimpanzees or gorillas. Thus, while other hominids have lost activity of this antiviral gene, it has been maintained, or re-acquired, as a more active antiviral gene in a subset of humans. Taken together, our results suggest that APOBEC3C is in fact involved in protecting hosts from lentiviruses. PMID:27732658

  12. Subcellular object quantification with Squassh3C and SquasshAnalyst.

    PubMed

    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.

  13. Subcellular object quantification with Squassh3C and SquasshAnalyst.

    PubMed

    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

  14. Capacitance of Ti3C2Tx MXene in ionic liquid electrolyte

    NASA Astrophysics Data System (ADS)

    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.

  15. Metagenomic chromosome conformation capture (meta3C) unveils the diversity of chromosome organization in microorganisms

    PubMed Central

    Marbouty, Martial; Cournac, Axel; Flot, Jean-François; Marie-Nelly, Hervé; Mozziconacci, Julien; Koszul, Romain

    2014-01-01

    Genomic analyses of microbial populations in their natural environment remain limited by the difficulty to assemble full genomes of individual species. Consequently, the chromosome organization of microorganisms has been investigated in a few model species, but the extent to which the features described can be generalized to other taxa remains unknown. Using controlled mixes of bacterial and yeast species, we developed meta3C, a metagenomic chromosome conformation capture approach that allows characterizing individual genomes and their average organization within a mix of organisms. Not only can meta3C be applied to species already sequenced, but a single meta3C library can be used for assembling, scaffolding and characterizing the tridimensional organization of unknown genomes. By applying meta3C to a semi-complex environmental sample, we confirmed its promising potential. Overall, this first meta3C study highlights the remarkable diversity of microorganisms chromosome organization, while providing an elegant and integrated approach to metagenomic analysis. DOI: http://dx.doi.org/10.7554/eLife.03318.001 PMID:25517076

  16. EBNA3C regulates p53 through induction of Aurora kinase B.

    PubMed

    Jha, Hem C; Yang, Karren; El-Naccache, Darine W; Sun, Zhiguo; Robertson, Erle S

    2015-03-20

    In multicellular organisms p53 maintains genomic integrity through activation of DNA repair, and apoptosis. EBNA3C can down regulate p53 transcriptional activity. Aurora kinase (AK) B phosphorylates p53, which leads to degradation of p53. Aberrant expression of AK-B is a hallmark of numerous human cancers. Therefore changes in the activities of p53 due to AK-B and EBNA3C expression is important for understanding EBV-mediated cell transformation. Here we show that the activities of p53 and its homolog p73 are dysregulated in EBV infected primary cells which can contribute to increased cell transformation. Further, we showed that the ETS-1 binding site is crucial for EBNA3C-mediated up-regulation of AK-B transcription. Further, we determined the Ser 215 residue of p53 is critical for functional regulation by AK-B and EBNA3C and that the kinase domain of AK-B which includes amino acid residues 106, 111 and 205 was important for p53 regulation. AK-B with a mutation at residue 207 was functionally similar to wild type AK-B in terms of its kinase activities and knockdown of AK-B led to enhanced p73 expression independent of p53. This study explores an additional mechanism by which p53 is regulated by AK-B and EBNA3C contributing to EBV-induced B-cell transformation.

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

    NASA Astrophysics Data System (ADS)

    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.

  18. Protease increases fermentation rate and ethanol yield in dry-grind ethanol production.

    PubMed

    Johnston, David B; McAloon, Andrew J

    2014-02-01

    The effects of acid protease and urea addition during the fermentation step were evaluated. The fermentations were also tested with and without the addition of urea to determine if protease altered the nitrogen requirements of the yeast. Results show that the addition of the protease had a statistically significant effect on the fermentation rate and yield. Fermentation rates and yields were improved with the addition of the protease over the corresponding controls without protease. Protease addition either with or with added urea resulted in a higher final ethanol yield than without the protease addition. Urea addition levels >1200 ppm of supplemental nitrogen inhibited ethanol production. The economic effects of the protease addition were evaluated by using process engineering and economic models developed at the Eastern Regional Research Center. The decrease in overall processing costs from protease addition was as high as $0.01/L (4 ¢/gal) of denatured ethanol produced.

  19. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    PubMed

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease. PMID:22888800

  20. The structure of a universally employed enzyme: V8 protease from Staphylococcus aureus

    SciTech Connect

    Prasad, Lata; Leduc, Yvonne; Hayakawa, Koto; Delbaere, Louis T.J.

    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.

  1. Expression, purification and molecular modeling of the NIa protease of Cardamom mosaic virus.

    PubMed

    Jebasingh, T; Pandaranayaka, Eswari P J; Mahalakshmi, A; Kasin Yadunandam, A; Krishnaswamy, S; Usha, R

    2013-01-01

    The NIa protease of Potyviridae is the major viral protease that processes potyviral polyproteins. The NIa protease coding region of Cardamom mosaic virus (CdMV) is amplified from the viral cDNA, cloned and expressed in Escherichia coli. NIa protease forms inclusion bodies in E.coli. The inclusion bodies are solubilized with 8 M urea, refolded and purified by Nickel-Nitrilotriacetic acid affinity chromatography. Three-dimensional modeling of the CdMV NIa protease is achieved by threading approach using the homologous X-ray crystallographic structure of Tobacco etch mosaic virus NIa protease. The model gave an insight in to the substrate specificities of the NIa proteases and predicted the complementation of nearby residues in the catalytic triad (H42, D74 and C141) mutants in the cis protease activity of CdMV NIa protease.

  2. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative.

    PubMed

    Bijina, B; Chellappan, Sreeja; Krishna, Jissa G; Basheer, Soorej M; Elyas, K K; Bahkali, Ali H; Chandrasekaran, M

    2011-07-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

  3. Protease inhibitor from Moringa oleifera with potential for use as therapeutic drug and as seafood preservative

    PubMed Central

    Bijina, B.; Chellappan, Sreeja; Krishna, Jissa G.; Basheer, Soorej M.; Elyas, K.K.; Bahkali, Ali H.; Chandrasekaran, M.

    2011-01-01

    Protease inhibitors are well known to have several applications in medicine and biotechnology. Several plant sources are known to return potential protease inhibitors. In this study plants belonging to different families of Leguminosae, Malvaceae, Rutaceae, Graminae and Moringaceae were screened for the protease inhibitor. Among them Moringa oleifera, belonging to the family Moringaceae, recorded high level of protease inhibitor activity after ammonium sulfate fractionation. M. oleifera, which grows throughout most of the tropics and having several industrial and medicinal uses, was selected as a source of protease inhibitor since so far no reports were made on isolation of the protease inhibitor. Among the different parts of M. oleifera tested, the crude extract isolated from the mature leaves and seeds showed the highest level of inhibition against trypsin. Among the various extraction media evaluated, the crude extract prepared in phosphate buffer showed maximum recovery of the protease inhibitor. The protease inhibitor recorded high inhibitory activity toward the serine proteases thrombin, elastase, chymotrypsin and the cysteine proteases cathepsin B and papain which have more importance in pharmaceutical industry. The protease inhibitor also showed complete inhibition of activities of the commercially available proteases of Bacillus licheniformis and Aspergillus oryzae. However, inhibitory activities toward subtilisin, esperase, pronase E and proteinase K were negligible. Further, it was found that the protease inhibitor could prevent proteolysis in a commercially valuable shrimp Penaeus monodon during storage indicating the scope for its application as a seafood preservative. This is the first report on isolation of a protease inhibitor from M. oleifera. PMID:23961135

  4. Kinetics of alkaline protease production by Streptomyces griseoflavus PTCC1130

    PubMed Central

    Hosseini, Seyed Vesal; Saffari, Zahra; Farhanghi, Ali; Atyabi, Seyed Mohammad; Norouzian, Dariush

    2016-01-01

    Background and Objectives: Proteases are a group of enzymes that catalyze the degradation of proteins resulting in the production of their amino acid constituents. They are the most important group of industrial enzymes which account for about 60% of total enzymes in the market and produced mainly by microorganisms. The attempts were made to study the kinetic parameters of protease produced by Streptomyces griseoflavus PTCC1130. Materials and Methods: Streptomyces griseoflavus PTCC1130 was grown on casein agar. Different media such as BM1, BM2, BM3 and BM4 were prepared. Data obtained from growth and protease production were subjected to kinetics evaluation. Casein was used as substrate for protease activity and the released soluble peptide bearing aromatic amino acid were quantified by Folin Cioclateaue reagent. Protein content of the enzyme and the sugar utilized by the organism were estimated by Bradford and Miller’s methods respectively. Results: Basal Medium named as BM1, BM2, BM3 and BM4(50 mL in 250 mL Erlen Meyer flasks) were screened out to evaluate protease production by Streptomyces griseoflavus PTCC1130. They were inoculated with known amount of seed culture and kept on rotary shaker. To obtain the specific growth rate, wet weight of biomass was plotted against the time. The clarified supernatant was used for the analysis of protease by measuring the soluble peptide containing aromatic amino acid residues employing Folin Cioclateaue reagent. Our results showed that maximum level of enzyme production (14035 U/L) was occurred at late exponential phase using Basal Medium supplemented with zinc sulfate (0.5g/L), casein (10g/L) at pH 6.5. Conclusions: A kinetic study of protease production by Streptomyces griseoflavus PTCC1130 provided highly quantitative information regarding the behavior of a system, which is essential to study the fermentation process. Exploitation of such kinetics analysis would be useful in commercialization of microbial enzyme

  5. Characterization of a chemostable serine alkaline protease from Periplaneta americana

    PubMed Central

    2013-01-01

    Background Proteases are important enzymes involved in numerous essential physiological processes and hold a strong potential for industrial applications. The proteolytic activity of insects’ gut is endowed by many isoforms with diverse properties and specificities. Thus, insect proteases can act as a tool in industrial processes. Results In the present study, purification and properties of a serine alkaline protease from Periplaneta americana and its potential application as an additive in various bio-formulations are reported. The enzyme was purified near to homogeneity by using acetone precipitation and Sephadex G-100 gel filtration chromatography. Enzyme activity was increased up to 4.2 fold after gel filtration chromatography. The purified enzyme appeared as single protein-band with a molecular mass of ~ 27.8 kDa in SDS-PAGE. The optimum pH and temperature for the proteolytic activity for purified protein were found around pH 8.0 and 60°C respectively. Complete inhibition of the purified enzyme by phenylmethylsulfonyl fluoride confirmed that the protease was of serine-type. The purified enzyme revealed high stability and compatibility towards detergents, oxidizing, reducing, and bleaching agents. In addition, enzyme also showed stability towards organic solvents and commercial detergents. Conclusion Several important properties of a serine protease from P. Americana were revealed. Moreover, insects can serve as excellent and alternative source of industrially important proteases with unique properties, which can be utilized as additives in detergents, stain removers and other bio-formulations. Properties of the P. americana protease accounted in the present investigation can be exploited further in various industrial processes. As an industrial prospective, identification of enzymes with varying essential properties from different insect species might be good approach and bioresource. PMID:24229392

  6. A Serine Protease Isolated from the Bristles of the Amazonic Caterpillar, Premolis semirufa, Is a Potent Complement System Activator

    PubMed Central

    Villas Boas, Isadora Maria; Pidde-Queiroz, Giselle; Magnoli, Fabio Carlos; Gonçalves-de-Andrade, Rute M.; van den Berg, Carmen W.; Tambourgi, Denise V.

    2015-01-01

    Background The caterpillar of the moth Premolis semirufa, commonly named pararama, is found in the Brazilian Amazon region. Accidental contact with the caterpillar bristles causes an intense itching sensation, followed by symptoms of an acute inflammation, which last for three to seven days after the first incident. After multiple accidents a chronic inflammatory reaction, called “Pararamose”, characterized by articular synovial membrane thickening with joint deformities common to chronic synovitis, frequently occurs. Although complement mediated inflammation may aid the host defense, inappropriate or excessive activation of the complement system and generation of anaphylatoxins can lead to inflammatory disorder and pathologies. The aim of the present study was to evaluate, in vitro, whether the Premolis semirufa’s bristles extract could interfere with the human complement system. Results The bristles extract was able to inhibit the haemolytic activity of the alternative pathway, as well as the activation of the lectin pathway, but had no effect on the classical pathway, and this inhibition seemed to be caused by activation and consumption of complement components. The extract induced the production of significant amounts of all three anaphylatoxins, C3a, C4a and C5a, promoted direct cleavage of C3, C4 and C5 and induced a significant generation of terminal complement complexes in normal human serum. By using molecular exclusion chromatography, a serine protease of 82 kDa, which activates complement, was isolated from P. semirufa bristles extract. The protease, named here as Ps82, reduced the haemolytic activity of the alternative and classical pathways and inhibited the lectin pathway. In addition, Ps82 induced the cleavage of C3, C4 and C5 and the generation of C3a and C4a in normal human serum and it was capable to cleave human purified C5 and generate C5a. The use of Phenanthroline, metalloprotease inhibitor, in the reactions did not significantly

  7. Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult.

    PubMed

    Mohamed, Saleh A; Fahmy, Afaf S; Mohamed, Tarek M; Hamdy, Soha M

    2005-10-01

    Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively. PMID:16102991

  8. Protease nexin-1 regulates retinal vascular development.

    PubMed

    Selbonne, Sonia; Francois, Deborah; Raoul, William; Boulaftali, Yacine; Sennlaub, Florian; Jandrot-Perrus, Martine; Bouton, Marie-Christine; Arocas, Véronique

    2015-10-01

    We recently identified protease nexin-1 (PN-1) or serpinE2, as a possibly underestimated player in maintaining angiogenic balance. Here, we used the well-characterized postnatal vascular development of newborn mouse retina to further investigate the role and the mechanism of action of PN-1 in physiological angiogenesis. The development of retinal vasculature was analysed by endothelial cell staining with isolectin B4. PN-1-deficient (PN-1(-/-)) retina displayed increased vascularization in the postnatal period, with elevated capillary thickness and density, compared to their wild-type littermate (WT). Moreover, PN-1(-/-) retina presented more veins/arteries than WT retina. The kinetics of retinal vasculature development, retinal VEGF expression and overall retinal structure were similar in WT and PN-1(-/-) mice, but we observed a hyperproliferation of vascular cells in PN-1(-/-) retina. Expression of PN-1 was analysed by immunoblotting and X-Gal staining of retinas from mice expressing beta-galactosidase under a PN-1 promoter. PN-1 was highly expressed in the first week following birth and then progressively decreased to a low level in adult retina where it localized on the retinal arteries. PCR arrays performed on mouse retinal RNA identified two angiogenesis-related factors, midkine and Smad5, that were overexpressed in PN-1(-/-) newborn mice and this was confirmed by RT-PCR. Both the higher vascularization and the overexpression of midkine and Smad5 mRNA were also observed in gastrocnemius muscle of PN-1(-/-) mice, suggesting that PN-1 interferes with these pathways. Together, our results demonstrate that PN-1 strongly limits physiological angiogenesis and suggest that modulation of PN-1 expression could represent a new way to regulate angiogenesis.

  9. Monocrystalline NbN nanofilms on a 3C-SiC/Si substrate

    NASA Astrophysics Data System (ADS)

    Gao, J. R.; Hajenius, M.; Tichelaar, F. D.; Klapwijk, T. M.; Voronov, B.; Grishin, E.; Gol'tsman, G.; Zorman, C. A.; Mehregany, M.

    2007-08-01

    The authors have realized NbN (100) nanofilms on a 3C-SiC (100)/Si(100) substrate by dc reactive magnetron sputtering at 800°C. High-resolution transmission electron microscopy (HRTEM) is used to characterize the films, showing a monocrystalline structure and confirming epitaxial growth on the 3C-SiC layer. A film ranging in thickness from 3.4to4.1nm shows a superconducting transition temperature of 11.8K, which is the highest reported for NbN films of comparable thickness. The NbN nano-films on 3C-SiC offer a promising alternative to improve terahertz detectors. For comparison, NbN nanofilms grown directly on Si substrates are also studied by HRTEM.

  10. Spitzer and near-infrared observations of the young supernova remnant 3C397

    NASA Astrophysics Data System (ADS)

    Rho, Jeonghee; Jarrett, Tom

    2016-06-01

    We present Spitzer IRS, IRAC and MIPS observations and near-infrared imaging and spectroscopy of the young supernova remnant 3C397 (G41.1-0.2). Near-infrared observations were made using the Palomar 200 inch telescope. Both mid- and near-infrared spectra are dominated by Fe lines and near-infrared imaging shows bright Fe emission with a shell-like morphology. There is no molecular hydrogen line belong to the SNR and some are in background. The Ni, Ar, S and Si lines are detected using IRS and hydrogen recombination lines are detected in near-infrared. Two nickel lines at 18.24 and 10.69 micron are detected. 3C397 is ejecta-dominated, and our observations support 3C397 to be a Type Ia supernova.

  11. Processing, compositional range, and mechanical behavior of the Mo(5)Si(3)C intermetallic compound

    NASA Astrophysics Data System (ADS)

    Ross, Eli Nickerson

    The intermetallic compound Mo5Si3C was studied in order to examine optimal processing methods, elevated temperature mechanical behavior, and the influence of crystal symmetry on the potential for improved room temperature ductility/toughness in ternary silicides. During the course of the investigation, samples were produced using nonconsumable arc melting and vacuum hot pressing of either blended or mechanically alloyed powders. The most significant challenge to the production of single-phase Mo5 Si3C was the significantly narrower range of homogeneity for the ternary compound than originally reported. The primary reason for this discrepancy is thought to be the use of x-ray diffraction (XRD) as the sole means of phase identification in the original study, whereas the current work augmented XRD with other microstructural analysis techniques. Further complicating the processing of Mo5Si3C was the occurrence of composition shifts during powder processing. These shifts in stoichiometry were correlated to observed microstructural features and likely the result of thermodynamically favored reactions between silica present in the starting powders and carbon. Because of these complications, materials for mechanical testing typically contained between one and 6 volume percent of phases other than Mo5Si3C, with samples having the nominal composition Mo4.8Si3C0.87 closest to single phase. An average microhardness value of 13.2 GPa was recorded for the ternary phase at room temperature, and an indentation fracture toughness of approximately 2.5 MPa·m1/2 was determined. In the temperature range 1000 to 1300°C, materials tested in compression displayed properties that were highly strain rate and grain size dependent, indicating the influence of boundary-controlled deformation mechanisms. The behavior of samples tested in four-point bending at 1200°C showed similar behavior, with larger grain size materials failing after limited deformation while finer grain size materials

  12. NGC 1068, 3C 273, and Scorpius X-1 - Circular polarization disputed.

    NASA Technical Reports Server (NTRS)

    Kemp, J. C.; Wolstencroft, R. D.; Swedlund, J. B.

    1972-01-01

    Discussion of data for the Seyfert galaxy NGC 1068, the quasar 3C 273, and the X-ray source Sco X-1, for all of which it has been claimed or strongly suggested that they have large circular polarization of the order of 1% (Severny et al., 1971). In contrast the present authors obtained unambiguously null results with upper limits about 0.05% for NGC 1068 and 3C 273. For Sco X-1 no statistically significant polarization was found on three nights, and only some marginal evidence for a fluctuation or transient polarization on one night.

  13. SATCOM antenna siting study on P-3C aircraft, volume 1

    NASA Technical Reports Server (NTRS)

    Bensman, D. A.; Marhefka, R. J.

    1991-01-01

    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.

  14. New Filling Technique and Performance Evaluations of the Cr3C2-C Peritectic Fixed Point

    NASA Astrophysics Data System (ADS)

    Sasajima, N.; Lowe, D.; Bai, C.; Yamada, Y.; Ara, C.

    2011-12-01

    The Cr3C2-C peritectic fixed point was investigated to test its capability to serve as a practical high-temperature fixed point. An improved filling technique where C/C sheet works as a wick and graphite paper as a hopper was applied successfully, and the long-term stability of the peritectic cell was evaluated by means of radiation thermometry. The repeatability of the melting point in one day was 7 mK with a melting range of approximately 100 mK. The cell was aged for 7 days, and the evaluated 56 melting temperatures during this period all fall within 90 mK, with a standard deviation of 19 mK. X-ray transmission photos showed that the ingot was filled uniformly in the crucible. After the evaluation of long-term stability, no clear degradation of the ingot shape and no leakage of molten metal were observed. From these results, it can be concluded that the Cr3C2-C peritectic cell has good stability and robustness, and the new filling technique was established. The impurity effect on the Cr3C2-C peritectic cell was also investigated by adding tungsten powder to another cell as the impurity component. After the observation of melting and freezing plateaux, the cell was cut in half to analyze the microstructure by means of electron probe microanalysis (EPMA) and laser ablation inductively coupled plasma mass spectrometer (LA-ICP-MS). The high concentration of impurity was observed in the area of the chromium-rich domain (eutectic mixture of Cr7C3 and Cr3C2), which suggests that impurities were rejected from the Cr3C2 peritectic phase during the peritectic freezing and were accumulated in the Cr7C3-Cr3C2 eutectic phase. This explains why the impurity effect is more severe for the Cr7C3-Cr3C2 eutectic point than for the Cr3C2-C peritectic point.

  15. SATCOM antenna siting study on P-3C aircraft, volume 1

    NASA Astrophysics Data System (ADS)

    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.

  16. The Hard X-Ray and Coordinated Multifrequency Properties of the Blazar 3C279

    NASA Technical Reports Server (NTRS)

    Collmar, W.; Boettcher, Markus

    2004-01-01

    Upon our successful AO-1 proposal no. 120085, the quasar 3C 279 has been observed in a coordinated multiwavelength campaign, including INTEGRAL, Chandra, and ground-based radio, near-infrared, and optical observations in June 2003. INTEGRAL observed 3C279 continuously for 300 ksec in INTEGRAL AO-1 between June 1 and June 5,2003. In addition, a Chandra pointing of 5 ksec was done on June 2, the Metsiihovi radio telescope contributed high-frequency radio observations, the SEST observed the source at mm wavelengths, and the 80 cm Turola telescope at La Palma and the 2.3 m telescope at Siding Springs, Australia contributed optical observations.

  17. Contribution of Gag and Protease to HIV-1 Phenotypic Drug Resistance in Pediatric Patients Failing Protease Inhibitor-Based Therapy

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Sutherland, Katherine; Parry, Chris M.; Cane, Patricia A.; Coovadia, Ashraf; Kuhn, Louise; Hunt, Gillian

    2016-01-01

    Protease inhibitors (PIs) are used as a first-line regimen in HIV-1-infected children. Here we investigated the phenotypic consequences of amino acid changes in Gag and protease on lopinavir (LPV) and ritonavir (RTV) susceptibility among pediatric patients failing PI therapy. The Gag-protease from isolates from 20 HIV-1 subtype C-infected pediatric patients failing an LPV and/or RTV-based regimen was phenotyped using a nonreplicative in vitro assay. Changes in sensitivity to LPV and RTV relative to that of the matched baseline (pretherapy) sample were calculated. Gag and protease amino acid substitutions associated with PI failure were created in a reference clone by site-directed mutagenesis and assessed. Predicted phenotypes were determined using the Stanford drug resistance algorithm. Phenotypic resistance or reduced susceptibility to RTV and/or LPV was observed in isolates from 10 (50%) patients, all of whom had been treated with RTV. In most cases, this was associated with protease resistance mutations, but substitutions at Gag cleavage and noncleavage sites were also detected. Gag amino acid substitutions were also found in isolates from three patients with reduced drug susceptibilities who had wild-type protease. Site-directed mutagenesis confirmed that some amino acid changes in Gag contributed to PI resistance but only in the presence of major protease resistance-associated substitutions. The isolates from all patients who received LPV exclusively were phenotypically susceptible. Baseline isolates from the 20 patients showed a large (47-fold) range in the 50% effective concentration of LPV, which accounted for most of the discordance seen between the experimentally determined and the predicted phenotypes. Overall, the inclusion of the gag gene and the use of matched baseline samples provided a more comprehensive assessment of the effect of PI-induced amino acid changes on PI resistance. The lack of phenotypic resistance to LPV supports the continued use of

  18. Regulation of Extracellular Protease Production in Bacillus cereus T: Characterization of Mutants Producing Altered Amounts of Protease

    PubMed Central

    Aronson, A. I.; Angelo, N.; Holt, S. C.

    1971-01-01

    Twenty-nine mutants of Bacillus cereus T were selected on casein agar for their inability to produce large amounts of extracellular protease. They all formed spores, and 27 were also auxotrophs for purines or pyrimidines. Upon reversion to prototrophy, a large fraction regained the capacity to produce protease. Conversely, reversion to normal protease production resulted in loss of the purine or pyrimidine requirement in a large fraction of the revertants. One spontaneous low-protease-producing pyrimidine auxotroph studied in detail grew as well as the wild type and produced spores which were identical to those produced by the wild type on the basis of heat resistance, dipicolinic acid content, density, and appearance in the electron microscope. The rate of protein turnover in the mutant was the same as the wild type. The mutant did grow poorly, however, when casein was the principal carbon source. A mutant excreting 5 to 10 times as much protease as the wild type was isolated as a secondary mutation from the hypoproducer discussed above. Loss of the pyrimidine requirement in this case did not alter the regulation of protease production. Although the secondary mutant grew somewhat faster in most media than the wild type, the final cell yield was lower. The spores of this mutant appeared to have excess coat on the basis of both electron microscopic and chemical studies. There appear to be closely related but distinct catabolic controls for both extracellular protease and spore formation. These controls can be dissociated as for the hypoproducers but can also appear integrated as for the hyperprotease producer. Images PMID:4104235

  19. Proteolytic Activation of the Protease-activated Receptor (PAR)-2 by the Glycosylphosphatidylinositol-anchored Serine Protease Testisin*

    PubMed Central

    Driesbaugh, Kathryn H.; Buzza, Marguerite S.; Martin, Erik W.; Conway, Gregory D.; Kao, Joseph P. Y.; Antalis, Toni M.

    2015-01-01

    Protease-activated receptors (PARs) are a family of seven-transmembrane, G-protein-coupled receptors that are activated by multiple serine proteases through specific N-terminal proteolytic cleavage and the unmasking of a tethered ligand. The majority of PAR-activating proteases described to date are soluble proteases that are active during injury, coagulation, and inflammation. Less investigation, however, has focused on the potential for membrane-anchored serine proteases to regulate PAR activation. Testisin is a unique trypsin-like serine protease that is tethered to the extracellular membrane of cells through a glycophosphatidylinositol (GPI) anchor. Here, we show that the N-terminal domain of PAR-2 is a substrate for testisin and that proteolytic cleavage of PAR-2 by recombinant testisin activates downstream signaling pathways, including intracellular Ca2+ mobilization and ERK1/2 phosphorylation. When testisin and PAR-2 are co-expressed in HeLa cells, GPI-anchored testisin specifically releases the PAR-2 tethered ligand. Conversely, knockdown of endogenous testisin in NCI/ADR-Res ovarian tumor cells reduces PAR-2 N-terminal proteolytic cleavage. The cleavage of PAR-2 by testisin induces activation of the intracellular serum-response element and NFκB signaling pathways and the induction of IL-8 and IL-6 cytokine gene expression. Furthermore, the activation of PAR-2 by testisin results in the loss and internalization of PAR-2 from the cell surface. This study reveals a new biological substrate for testisin and is the first demonstration of the activation of a PAR by a serine protease GPI-linked to the cell surface. PMID:25519908

  20. Diversity of both the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China sea.

    PubMed

    Zhou, Ming-Yang; Chen, Xiu-Lan; Zhao, Hui-Lin; Dang, Hong-Yue; Luan, Xi-Wu; Zhang, Xi-Ying; He, Hai-Lun; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2009-10-01

    Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

  1. Protease Inhibitors in View of Peptide Substrate Databases

    PubMed Central

    2016-01-01

    Protease substrate profiling has nowadays almost become a routine task for experimentalists, and the knowledge on protease peptide substrates is easily accessible via the MEROPS database. We present a shape-based virtual screening workflow using vROCS that applies the information about the specificity of the proteases to find new small-molecule inhibitors. Peptide substrate sequences for three to four substrate positions of each substrate from the MEROPS database were used to build the training set. Two-dimensional substrate sequences were converted to three-dimensional conformations through mutation of a template peptide substrate. The vROCS query was built from single amino acid queries for each substrate position considering the relative frequencies of the amino acids. The peptide-substrate-based shape-based virtual screening approach gives good performance for the four proteases thrombin, factor Xa, factor VIIa, and caspase-3 with the DUD-E data set. The results show that the method works for protease targets with different specificity profiles as well as for targets with different active-site mechanisms. As no structure of the target and no information on small-molecule inhibitors are required to use our approach, the method has significant advantages in comparison with conventional structure- and ligand-based methods. PMID:27247997

  2. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    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.

  3. Mitochondrial cereblon functions as a Lon-type protease.

    PubMed

    Kataoka, Kosuke; Nakamura, China; Asahi, Toru; Sawamura, Naoya

    2016-07-15

    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.

  4. Emerging roles for diverse intramembrane proteases in plant biology.

    PubMed

    Adam, Zach

    2013-12-01

    Progress in the field of regulated intramembrane proteolysis (RIP) in recent years has made its impact on plant biology as well. Although this field within plant research is still in its infancy, some interesting observations have started to emerge. Gene encoding orthologs of rhomboid proteases, site-2 proteases (S2P), presenilin/γ-secretases, and signal peptide peptidases are found in plant genomes and some of these gene products were identified in different plant cell membranes. The lack of chloroplast-located rhomboid proteases was associated with reduced fertility and aberrations in flower morphology. Mutations in homologues of S2P resulted in chlorophyll deficiency and impaired chloroplast development. An S2P was also implicated in the response to ER stress through cleavage of ER-membrane bZIP transcription factors, allowing their migration to the nucleus and activation of the transcription of BiP chaperones. Other membrane-bound transcription factors of the NAC and PHD families were also demonstrated to undergo RIP and relocalization to the nucleus. These and other new data are expected to shed more light on the roles of intramembrane proteases in plant biology in the future. This article is part of a Special Issue entitled: Intramembrane Proteases.

  5. Alkaline protease from Thermoactinomyces sp. RS1 mitigates industrial pollution.

    PubMed

    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.

  6. Extracellular proteases are released by ciliates in defined seawater microcosms.

    PubMed

    Thao, Ngo Vy; Nozawa, Akino; Obayashi, Yumiko; Kitamura, Shin-Ichi; Yokokawa, Taichi; Suzuki, Satoru

    2015-08-01

    The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns.

  7. Plant collagenase: unique collagenolytic activity of cysteine proteases from ginger.

    PubMed

    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

  8. Characterization, biomedical and agricultural applications of protease inhibitors: A review.

    PubMed

    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

  9. Predictors of virologic response to ritonavir-boosted protease inhibitors.

    PubMed

    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

  10. Extracellular proteases are released by ciliates in defined seawater microcosms.

    PubMed

    Thao, Ngo Vy; Nozawa, Akino; Obayashi, Yumiko; Kitamura, Shin-Ichi; Yokokawa, Taichi; Suzuki, Satoru

    2015-08-01

    The biodegradation of proteins in seawater requires various proteases which are commonly thought to be mainly derived from heterotrophic bacteria. We, however, found that protists showed a high protease activity and continuously produced trypsin-type enzymes. The free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium was isolated and used for microcosm incubation with different concentrations of killed bacteria as food for 10 days. The results showed that the co-existence of the ciliate with its associated bacterium produced a significant protease activity in both cell-associated and cell-free fractions while that in the associated bacterium only microcosm was negligible. The protease profiles are different between cell-associated and cell-free fractions, and a trypsin-type enzyme hydrolyzing Boc-Val-Leu-Lys-MCA was detected throughout the period in the presence of ciliates. This suggests that ciliates release proteases into the surrounding environment which could play a role in protein digestion outside cells. It has been previously suggested that bacteria are the major transformers in seawater. We here present additional data which indicates that protists, or at least ciliates with their specific enzymes, are a potential player in organic matter degradation in water columns. PMID:26115436

  11. Three Pseudomonas aeruginosa strains with different protease profiles.

    PubMed

    Andrejko, Mariola; Zdybicka-Barabas, Agnieszka; Janczarek, Monika; Cytryńska, Małgorzata

    2013-01-01

    The proteolytic activity of three Pseudomonas aeruginosa strains, ATCC 27853 - a reference strain, and two clinical isolates was tested. The activity was examined after culturing the bacteria in two different growth media: the minimal M9 medium and rich Luria-Bertani broth (LB). Based on zymograms and protease activity specific assays, it was concluded that the reference strain produced three proteolytic enzymes in the LB medium: protease IV, elastase B and elastase A, while alkaline protease was only produced in the M9 medium. The clinical isolates of P. aeruginosa produced elastase B and alkaline protease when grown in the LB medium and the minimal M9 medium, respectively. PCR analysis confirmed the presence of both the lasB gene encoding elastase B and aprA coding for alkaline protease in the genomes of the three P. aeruginosa strains analyzed. The expression of these genes coding for two important P. aeruginosa virulence factors was dependent on the growth conditions in all the strains studied. The contribution of the extracellular proteinases to the virulence of P. aeruginosa strains used in this study was investigated using an insect model, the greater wax moth Galleria mellonella.

  12. Hydrophobic Core Flexibility Modulates Enzyme Activity in HIV-1 Protease

    SciTech Connect

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

    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.

  13. Mitochondrial cereblon functions as a Lon-type protease

    PubMed Central

    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

  14. Selection of multiple human immunodeficiency virus type 1 variants that encode viral proteases with decreased sensitivity to an inhibitor of the viral protease.

    PubMed Central

    Kaplan, A H; Michael, S F; Wehbie, R S; Knigge, M F; Paul, D A; Everitt, L; Kempf, D J; Norbeck, D W; Erickson, J W; Swanstrom, R

    1994-01-01

    Inhibitors of the human immunodeficiency virus type 1 (HIV-1) protease represent a promising addition to the available agents used to inhibit virus replication in a therapeutic setting. HIV-1 is capable of generating phenotypic variants in the face of a variety of selective pressures. The potential to generate variants with reduced sensitivity to a protease inhibitor was examined by selecting for virus growth in cell culture in the presence of the protease inhibitor A-77003. Virus variants grew out in the presence of the inhibitor, and these variants encoded proteases with reduced sensitivity to the inhibitor. Variants were identified that encoded changes in each of the three subsites of the protease that interact with the inhibitor. HIV-1 displays significant potential for altering its interaction with this protease inhibitor, suggesting the need for multiple protease inhibitors with varying specificities. Images PMID:8202533

  15. Jet-induced star formation in 3C 285 and Minkowski's Object

    NASA Astrophysics Data System (ADS)

    Salomé, Q.; Salomé, P.; Combes, F.

    2015-02-01

    How efficiently star formation proceeds in galaxies is still an open question. Recent studies suggest that active galactic nucleus (AGN) can regulate the gas accretion and thus slow down star formation (negative feedback). However, evidence of AGN positive feedback has also been observed in a few radio galaxies (e.g. Centaurus A, Minkowski's Object, 3C 285, and the higher redshift 4C 41.17). Here we present CO observations of 3C 285 and Minkowski's Object, which are examples of jet-induced star formation. A spot (named 3C 285/09.6 in the present paper) aligned with the 3C 285 radio jet at a projected distance of ~70 kpc from the galaxy centre shows star formation that is detected in optical emission. Minkowski's Object is located along the jet of NGC 541 and also shows star formation. Knowing the distribution of molecular gas along the jets is a way to study the physical processes at play in the AGN interaction with the intergalactic medium. We observed CO lines in 3C 285, NGC 541, 3C 285/09.6, and Minkowski's Object with the IRAM 30 m telescope. In the central galaxies, the spectra present a double-horn profile, typical of a rotation pattern, from which we are able to estimate the molecular gas density profile of the galaxy. The molecular gas appears to be in a compact reservoir, which could be evidence of an early phase of the gas accretion after a recent merger event in 3C 285. No kinematic signature of a molecular outflow is detected by the 30 m telescope. Interestingly, 3C 285/09.6 and Minkowski's Object are not detected in CO. The cold gas mass upper limits are consistent with a star formation induced by the compression of dense ambient material by the jet. The depletion time scales in 3C 285/09.6 and Minkowski's Object are of the order of and even shorter than what is found in 3C 285, NGC 541, and local spiral galaxies (109 yr). The upper limit of the molecular gas surface density in 3C 285/09.6 at least follows a Schmidt-Kennicutt law if the emitting region

  16. Anti-Enterovirus 71 Effects of Chrysin and Its Phosphate Ester

    PubMed Central

    Du, Jiang; Cui, Sheng; Yang, Fan; Jin, Qi

    2014-01-01

    Enterovirus 71 (EV71) can cause severe disease and even lead to death in children, and an effective antiviral drug is currently unavailable. The anti-EV71 effect of chrysin (5,7-dihydroxyflavone), a natural flavonoid commonly found in many plants, was tested in this report. By using the predicting program Autodock 4.0 and an in vitro protease inhibition assay, we found that chrysin could suppress viral 3Cpro activity. Replication of viral RNA and production of viral capsid protein and the infectious virion were strongly inhibited by chrysin, without noticeable cytotoxicity. Cytopathic effects on cells were also prevented. Diisopropyl chrysin-7-yl phosphate (CPI), the phosphate ester for chrysin, was generated through a simplified Atheron-Todd reaction to achieve stronger anti-viral activity. CPI was also able to bind with and inhibit viral 3Cpro activity in vitro. As expected, CPI demonstrated more potent antiviral activity against EV71. PMID:24598537

  17. Diversity of cultivable protease-producing bacteria in sediments of Jiaozhou Bay, China

    PubMed Central

    Zhang, Xi-Ying; Han, Xiao-Xu; Chen, Xiu-Lan; Dang, Hong-Yue; Xie, Bin-Bin; Qin, Qi-Long; Shi, Mei; Zhou, Bai-Cheng; Zhang, Yu-Zhong

    2015-01-01

    Although protease-producing bacteria are key players in the degradation of organic nitrogen and essential for the nitrogen recycling in marine sediments, diversity of both these bacteria and their extracellular proteases is still largely unknown. This study investigated the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the eutrophied Jiaozhou Bay, China through phylogenetic analysis and protease inhibitor tests. The abundance of the cultivable protease-producing bacteria was up to 104 cells/g in all six sediment samples. The cultivated protease-producing bacteria mostly belonged to the phyla Proteobacteria and Firmicutes with the predominant genera being Photobacterium (39.4%), Bacillus (25.8%), and Vibrio (19.7%). Protease inhibitor tests revealed that extracellular proteases secreted by the bacteria were mainly serine proteases and/or metalloproteases with relatively low proportions of cysteine proteases. This study represents the first comprehensive analysis on the diversity of protease-producing bacteria and their extracellular proteases in sediments of a eutrophic bay. PMID:26441943

  18. Evidence for Reduced Drug Susceptibility without Emergence of Major Protease Mutations following Protease Inhibitor Monotherapy Failure in the SARA Trial

    PubMed Central

    Sutherland, Katherine A.; Parry, Chris M.; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Gilks, Charles F.; Goodall, Ruth; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Gupta, Ravindra K.

    2015-01-01

    Background Major protease mutations are rarely observed following failure with protease inhibitors (PI), and other viral determinants of failure to PI are poorly understood. We therefore characterized Gag-Protease phenotypic susceptibility in subtype A and D viruses circulating in East Africa following viral rebound on PIs. Methods Samples from baseline and treatment failure in patients enrolled in the second line LPV/r trial SARA underwent phenotypic susceptibility testing. Data were expressed as fold-change in susceptibility relative to a LPV-susceptible reference strain. Results We cloned 48 Gag-Protease containing sequences from seven individuals and performed drug resistance phenotyping from pre-PI and treatment failure timepoints in seven patients. For the six patients where major protease inhibitor resistance mutations did not emerge, mean fold-change EC50 to LPV was 4.07 fold (95% CI, 2.08–6.07) at the pre-PI timepoint. Following viral failure the mean fold-change in EC50 to LPV was 4.25 fold (95% CI, 1.39–7.11, p = 0.91). All viruses remained susceptible to DRV. In our assay system, the major PI resistance mutation I84V, which emerged in one individual, conferred a 10.5-fold reduction in LPV susceptibility. One of the six patients exhibited a significant reduction in susceptibility between pre-PI and failure timepoints (from 4.7 fold to 9.6 fold) in the absence of known major mutations in protease, but associated with changes in Gag: V7I, G49D, R69Q, A120D, Q127K, N375S and I462S. Phylogenetic analysis provided evidence of the emergence of genetically distinct viruses at the time of treatment failure, indicating ongoing viral evolution in Gag-protease under PI pressure. Conclusions Here we observe in one patient the development of significantly reduced susceptibility conferred by changes in Gag which may have contributed to treatment failure on a protease inhibitor containing regimen. Further phenotype-genotype studies are required to elucidate genetic

  19. The singular gas-phase structure of 1-aminocyclopropanecarboxylic acid (Ac3c).

    PubMed

    Jiménez, Ana I; Vaquero, Vanesa; Cabezas, Carlos; López, Juan C; Cativiela, Carlos; Alonso, José L

    2011-07-13

    The natural nonproteinogenic α-amino acid 1-aminocyclopropanecarboxylic acid (Ac(3)c) has been vaporized by laser ablation and studied in the gas phase by molecular-beam Fourier transform microwave spectroscopy. Comparison of the experimental rotational and (14)N nuclear quadrupole coupling constants with the values predicted ab initio for these parameters has allowed the unambiguous identification of three Ac(3)c conformers differing in the hydrogen bonding pattern. Two of them resemble those characterized before for the coded aliphatic α-amino acids. Remarkably, a third conformer predicted to be energetically accessible for all of these amino acids but never observed (the so-called "missing conformer") has been found for Ac(3)c, close in energy to the global minimum. This is the first time that such a conformer, stabilized by an N-H···O(H) hydrogen bond, is detected in the rotational spectrum of a gaseous α-amino acid with a nonpolar side chain. The conjugative interaction established between the cyclopropane ring and the adjacent carbonyl group seems to be responsible for the unique conformational properties exhibited by Ac(3)c.

  20. Ti3C2Tx Filler Effect on the Proton Conduction Property of Polymer Electrolyte Membrane.

    PubMed

    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

  1. PIK3C2B inhibition improves function and prolongs survival in myotubular myopathy animal models.

    PubMed

    Sabha, Nesrin; Volpatti, Jonathan R; Gonorazky, Hernan; Reifler, Aaron; Davidson, Ann E; Li, Xingli; Eltayeb, Nadine M; Dall'Armi, Claudia; Di Paolo, Gilbert; Brooks, Susan V; Buj-Bello, Ana; Feldman, Eva L; Dowling, James J

    2016-09-01

    Myotubular myopathy (MTM) is a devastating pediatric neuromuscular disorder of phosphoinositide (PIP) metabolism resulting from mutations of the PIP phosphatase MTM1 for which there are no treatments. We have previously shown phosphatidylinositol-3-phosphate (PI3P) accumulation in animal models of MTM. Here, we tested the hypothesis that lowering PI3P levels may prevent or reverse the MTM disease process. To test this, we targeted class II and III PI3 kinases (PI3Ks) in an MTM1-deficient mouse model. Muscle-specific ablation of Pik3c2b, but not Pik3c3, resulted in complete prevention of the MTM phenotype, and postsymptomatic targeting promoted a striking rescue of disease. We confirmed this genetic interaction in zebrafish, and additionally showed that certain PI3K inhibitors prevented development of the zebrafish mtm phenotype. Finally, the PI3K inhibitor wortmannin improved motor function and prolonged lifespan of the Mtm1-deficient mice. In all, we have identified Pik3c2b as a genetic modifier of Mtm1 mutation and demonstrated that PIK3C2B inhibition is a potential treatment strategy for MTM. In addition, we set the groundwork for similar reciprocal inhibition approaches for treating other PIP metabolic disorders and highlight the importance of modifier gene pathways as therapeutic targets. PMID:27548528

  2. An increase of Optical Activity in the Quasar 3C454.3

    NASA Astrophysics Data System (ADS)

    Jorstad, Svetlana

    2016-06-01

    The quasar 3C454.3 shows an increase of the optical activity during the last three nights. According to our observations at the Perkins telescope of the Lowell Observatory (Flagstaff, AZ) the brightness of the source on June 10 (JD 2457549.8770) in R band was 15.817+-0.018 with a of polarization of 2.27+-0.39%.

  3. 17 CFR 270.3c-5 - Beneficial ownership by knowledgeable employees and certain other persons.

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... SECURITIES AND EXCHANGE COMMISSION (CONTINUED) RULES AND REGULATIONS, INVESTMENT COMPANY ACT OF 1940 § 270.3c... section 2(a)(3) of the Act , that manages the investment activities of a Covered Company. For purposes of this definition, the term “investment company” as used in section 2(a)(3) of the Act includes a...

  4. 40 CFR 152.112 - Approval of registration under FIFRA sec. 3(c)(5).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 40 Protection of Environment 24 2011-07-01 2011-07-01 false Approval of registration under FIFRA sec. 3(c)(5). 152.112 Section 152.112 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... to, evidence of compliance with subpart E of this part; (b) The Agency has reviewed all relevant...

  5. Ti3C2Tx Filler Effect on the Proton Conduction Property of Polymer Electrolyte Membrane.

    PubMed

    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.

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

    PubMed

    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

  7. 40 CFR 59.1 - Final determinations under Section 183(e)(3)(C) of the CAA.

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 40 Protection of Environment 5 2010-07-01 2010-07-01 false Final determinations under Section 183(e)(3)(C) of the CAA. 59.1 Section 59.1 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) NATIONAL VOLATILE ORGANIC COMPOUND EMISSION STANDARDS FOR CONSUMER...

  8. Structural Basis for Molecular Discrimination by a 3',3'-cGAMP Sensing Riboswitch

    DOE PAGES

    Ren, Aiming; Wang, Xin  C.; Kellenberger, Colleen  A.; Rajashankar, Kanagalaghatta  R.; Jones, Roger  A.; Hammond, Ming  C.; Patel, Dinshaw  J.

    2015-04-07

    Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate the innate immune response in mammals. Besides protein receptors, there are bacterial riboswitches that selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch that targets 3',3'-cGAMP, which is distinguished from the endogenous mammalian signal 2',3'-cGAMP by its backbone connectivity. Here, we report on structures of the aptamer domain of the 3',3'-cGAMP riboswitch from Geobacter in the 3',3'-cGAMP and c-di-GMP bound states. The riboswitch adopts a tuning forklike architecture with a junctional ligand-binding pocket and different orientations of the arms are correlated with the identity of the boundmore » cyclic dinucleotide. Subsequent biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, which has implications for both the assignment and reengineering of riboswitches in this structural class.« less

  9. Fast optical brightening of the blazar 3C 454.3 (2251+158)

    NASA Astrophysics Data System (ADS)

    Mirzaqulov, D. O.; Ehgamberdiev, Sh. A.; Villata, M.; Raiteri, C. M.

    2013-09-01

    With reference to ATel #5411, the GLAST-AGILE Support Program (GASP) of the Whole Earth Blazar Telescope (WEBT) reports on the recent observation of a strong optical brightening of the gamma-loud quasar 3C 454.3. This is one of the 28 blazars for which the GASP performs a long-term, multiwavelength monitoring.

  10. Identification of γ-ray emission from 3C 345 and NRAO 512

    NASA Astrophysics Data System (ADS)

    Schinzel, F. K.; Sokolovsky, K. V.; 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.; Bravo Calle, O. J. A.; Carosati, D.; Casas, R.; Gómez, 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.

    2011-08-01

    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 γ-ray energies ≥ 20 MeV. Recently a bright γ-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 γ-ray emitting counterpart of 1FGL J1642.5+3947. The source 3C 345 is identified as the main contributor for this γ-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 increasing γ-ray activity, possibly adding flux to 1FGL J1642.5+3947. For the same time period and during the summer of 2010, an increase of radio, optical and X-ray activity of NRAO 512 was observed. No γ-ray emission from B3 1640 + 396 was detected.

  11. Distance and age of the pulsar wind nebula 3C 58

    NASA Astrophysics Data System (ADS)

    Kothes, R.

    2013-12-01

    Context. A growing number of researchers present evidence that the pulsar wind nebula 3C 58 is much older than predicted by its proposed connection to the historical supernova of A.D. 1181. There is also a great diversity of arguments. The strongest of these arguments rely heavily on the assumed distance of 3.2 kpc determined with H i absorption measurements. Aims: This publication aims at determining a more accurate distance for 3C 58 and at re-evaluating the arguments for an older age. Methods: I have re-visited the distance determination of 3C 58 based on new H i data from the Canadian Galactic Plane Survey and our recent improvements in the knowledge of the rotation curve of the outer Milky Way Galaxy. I also used newly determined distances to objects in the neighbourhood, which are based on direct measurements by trigonometric parallax. Results: I have derived a new more reliable distance estimate of 2 kpc for 3C 58. This makes the connection between the pulsar wind nebula and the historical event from A.D. 1181 once again much more viable. With permission of Crown Copyright 2013 NRC Canada.

  12. Renewed gamma-ray activity of the Blazar 3C 454.3 detected by AGILE

    NASA Astrophysics Data System (ADS)

    Bulgarelli, A.; Parmiggiani, N.; Fioretti, V.; Zoli, A.; Lucarelli, F.; Verrecchia, F.; Pittori, C.; Vercellone, S.; Piano, G.; Munar-Adrover, P.; Tavani, M.; Donnarumma, I.; Striani, E.; Cardillo, M.; Gianotti, F.; Trifoglio, M.; Giuliani, A.; Mereghetti, S.; Caraveo, P.; Perotti, F.; Chen, A.; Argan, A.; Costa, E.; Del Monte, E.; Evangelista, Y.; Feroci, M.; Lazzarotto, F.; Lapshov, I.; Pacciani, L.; Soffitta, P.; Sabatini, S.; Vittorini, V.; Pucella, G.; Rapisarda, M.; Di Cocco, G.; Fuschino, F.; Galli, M.; Labanti, C.; Marisaldi, M.; Pellizzoni, A.; Pilia, M.; Trois, A.; Barbiellini, G.; Vallazza, E.; Longo, F.; Morselli, A.; Picozza, P.; Prest, M.; Lipari, P.; Zanello, D.; Cattaneo, P. W.; Rappoldi, A.; Colafrancesco, S.; Ferrari, A.; Antonelli, A.; Giommi, P.; Salotti, L.; Valentini, G.; D'Amico, F.

    2016-06-01

    The AGILE satellite is detecting a significant enhancement in gamma-ray activity from the FSRQ 3C 454.3 (known as 1AGLR J2254+1609) since the recent AGILE ATel #9157, and the optical activity reported in ATel #9150.

  13. Finite-Element Modeling of 3C-SiC Membranes

    NASA Technical Reports Server (NTRS)

    DeAnna, R. G.; Mitchell, J.; Zorman, C. A.; Mehregany, M.

    2000-01-01

    Finite-element modeling (FEM) of 3C-SiC thin-film membranes on Si substrates was used to determine the residual stress and center deflection with applied pressure. The anisotropic, three-dimensional model includes the entire 3C-SiC membrane and Si substrate with appropriate material properties and boundary conditions. Residual stress due to the thermal-expansion-coefficient mismatch between the3C-SiC film and Si substrate was included in the model. Both before-and after-etching, residual stresses were calculated. In-plane membrane stress and normal deflection with applied pressure were also calculated. FEM results predict a tensile residual stress fo 259 MPa in the 3C-SiC membrane before etching. This decreases to 247 MPa after etching the substrate below the membrane. The residual stress experimentally measured on sample made at Case Western Reserve University was 280 MPa on post-etched membranes. This is excellent agreement when an additional 30-40 MPa of residual stress to account for lattice mismatch is added to the FEM results.

  14. 40 CFR 152.112 - Approval of registration under FIFRA sec. 3(c)(5).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 25 2013-07-01 2013-07-01 false Approval of registration under FIFRA sec. 3(c)(5). 152.112 Section 152.112 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... effects on the environment, and that, when used in accordance with widespread and commonly...

  15. 40 CFR 152.112 - Approval of registration under FIFRA sec. 3(c)(5).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 40 Protection of Environment 25 2012-07-01 2012-07-01 false Approval of registration under FIFRA sec. 3(c)(5). 152.112 Section 152.112 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY... effects on the environment, and that, when used in accordance with widespread and commonly...

  16. Semipolar (202̅3) nitrides grown on 3C-SiC/(001) Si substrates

    NASA Astrophysics Data System (ADS)

    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).

  17. 17 CFR 270.3c-5 - Beneficial ownership by knowledgeable employees and certain other persons.

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... unit, division or function (such as sales, administration or finance), any other officer who performs a... with respect to any Covered Company means any natural person who is: (i) An Executive Officer, director... for the exclusion provided by section 3(c)(7) of the Act . (b) For purposes of determining the...

  18. 32 CFR 989.26 - Classified actions (40 CFR 1507.3(c)).

    Code of Federal Regulations, 2010 CFR

    2010-07-01

    ... 32 National Defense 6 2010-07-01 2010-07-01 false Classified actions (40 CFR 1507.3(c)). 989.26... ENVIRONMENTAL PROTECTION ENVIRONMENTAL IMPACT ANALYSIS PROCESS (EIAP) § 989.26 Classified actions (40 CFR 1507.3... classified, the EPF must make a report of the matter to SAF/IEE, including proposed modifications of...

  19. 32 CFR 989.26 - Classified actions (40 CFR 1507.3(c)).

    Code of Federal Regulations, 2012 CFR

    2012-07-01

    ... 32 National Defense 6 2012-07-01 2012-07-01 false Classified actions (40 CFR 1507.3(c)). 989.26... ENVIRONMENTAL PROTECTION ENVIRONMENTAL IMPACT ANALYSIS PROCESS (EIAP) § 989.26 Classified actions (40 CFR 1507.3... classified, the EPF must make a report of the matter to SAF/IEE, including proposed modifications of...

  20. 32 CFR 989.26 - Classified actions (40 CFR 1507.3(c)).

    Code of Federal Regulations, 2011 CFR

    2011-07-01

    ... 32 National Defense 6 2011-07-01 2011-07-01 false Classified actions (40 CFR 1507.3(c)). 989.26... ENVIRONMENTAL PROTECTION ENVIRONMENTAL IMPACT ANALYSIS PROCESS (EIAP) § 989.26 Classified actions (40 CFR 1507.3... classified, the EPF must make a report of the matter to SAF/IEE, including proposed modifications of...

  1. 32 CFR 989.26 - Classified actions (40 CFR 1507.3(c)).

    Code of Federal Regulations, 2014 CFR

    2014-07-01

    ... 32 National Defense 6 2014-07-01 2014-07-01 false Classified actions (40 CFR 1507.3(c)). 989.26... ENVIRONMENTAL PROTECTION ENVIRONMENTAL IMPACT ANALYSIS PROCESS (EIAP) § 989.26 Classified actions (40 CFR 1507.3... classified, the EPF must make a report of the matter to SAF/IEI, including proposed modifications of...

  2. 32 CFR 989.26 - Classified actions (40 CFR 1507.3(c)).

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 32 National Defense 6 2013-07-01 2013-07-01 false Classified actions (40 CFR 1507.3(c)). 989.26... ENVIRONMENTAL PROTECTION ENVIRONMENTAL IMPACT ANALYSIS PROCESS (EIAP) § 989.26 Classified actions (40 CFR 1507.3... classified, the EPF must make a report of the matter to SAF/IEE, including proposed modifications of...

  3. Chandra Detection of a Parsec Scale Wind in the Broad Line Radio Galaxy 3C 382

    NASA Technical Reports Server (NTRS)

    Reeves, J. N.; Sambruna, R. M.; Braito, V.; Eracleous, Michael

    2009-01-01

    We present unambiguous evidence for a parsec scale wind in the Broad-Line Radio Galaxy (BLRG) 3C 382, the first radio-loud AGN whereby an outflow has been measured with X-ray grating spectroscopy. A 118 ks Chandra grating (HETG) observation of 3C 382 has revealed the presence of several high ionization absorption lines in the soft X-ray band, from Fe, Ne, Mg and Si. The absorption lines are blue-shifted with respect to the systemic velocity of 3C 382 by -840+/-60 km/s and are resolved by Chandra with a velocity width of sigma = 340+/-70 km/s. The outflow appears to originate from a single zone of gas of column density N(sub H) = 1.3 x 10(exp 21)/sq cm and ionization parameter log(E/erg/cm/s) = 2.45. From the above measurements we calculate that the outflow is observed on parsec scales, within the likely range from 10-1000 pc, i.e., consistent with an origin in the Narrow Line Region. Finally we also discuss the possibility of a much faster (0.1c) outflow component, based on a blue-shifted iron K(alpha) emission line in the Suzaku observation of 3C 382, which could have an origin in an accretion disk wind.

  4. Structural basis for molecular discrimination by a 3',3'-cGAMP sensing riboswitch.

    PubMed

    Ren, Aiming; Wang, Xin C; Kellenberger, Colleen A; Rajashankar, Kanagalaghatta R; Jones, Roger A; Hammond, Ming C; Patel, Dinshaw J

    2015-04-01

    Cyclic dinucleotides are second messengers that target the adaptor STING and stimulate the innate immune response in mammals. Besides protein receptors, there are bacterial riboswitches that selectively recognize cyclic dinucleotides. We recently discovered a natural riboswitch that targets 3',3'-cGAMP, which is distinguished from the endogenous mammalian signal 2',3'-cGAMP by its backbone connectivity. Here, we report on structures of the aptamer domain of the 3',3'-cGAMP riboswitch from Geobacter in the 3',3'-cGAMP and c-di-GMP bound states. The riboswitch adopts a tuning fork-like architecture with a junctional ligand-binding pocket and different orientations of the arms are correlated with the identity of the bound cyclic dinucleotide. Subsequent biochemical experiments revealed that specificity of ligand recognition can be affected by point mutations outside of the binding pocket, which has implications for both the assignment and reengineering of riboswitches in this structural class. PMID:25818298

  5. Renewed gamma-ray activity of the Blazar 3C 454.3 detected by AGILE

    NASA Astrophysics Data System (ADS)

    Verrecchia, F.; Fioretti, V.; Lucarelli, F.; Pittori, C.; Bulgarelli, A.; Tavani, M.; Vercellone, S.; Piano, G.; Donnarumma, I.; Striani, E.; Cardillo, M.; Gianotti, F.; Trifoglio, M.; Giuliani, A.; Mereghetti, S.; Caraveo, P.; Perotti, F.; Chen, A.; Colafrancesco, S.; Del Monte, E.; Evangelista, Y.; Feroci, M.; Lazzarotto, F.; Pacciani, L.; Soffitta, P.; Costa, E.; Lapshov, I.; Rapisarda, M.; Argan, A.; Pucella, G.; Sabatini, S.; Trois, A.; Vittorini, V.; Fuschino, F.; Galli, M.; Labanti, C.; Marisaldi, M.; Di Cocco, G.; Pellizzoni, A.; Pilia, M.; Barbiellini, G.; Vallazza, E.; Longo, F.; Morselli, A.; Picozza, P.; Prest, M.; Lipari, P.; Zanello, D.; Cattaneo, P. W.; Rappoldi, A.; Giommi, P.; Salotti, L.; Valentini, G.

    2014-06-01

    The AGILE satellite detects a significant enhancement in gamma-ray activity from the FSRQ 3C 454.3 (known as 1AGLR J2254+1609 and 2FGL J2253.9+1609) since the recent AGILE ATel #6182, and the following NIR flare reported by Carrasco et al. ...

  6. High-Velocity Gas in the SN1991T/3C273 Sightline

    NASA Astrophysics Data System (ADS)

    Meyer, David M.

    Meyer & Roth (1991) have recently obtained high SIN observations of the Ca II absorption toward SN1991T and 3C273 which exhibit weak components at vLSR=-87, +215, and +263 km s^-1 toward SN1991T that are absent in the adjacent (sep.=1.4degrees) 3C273 sightline. In this proposal, we discuss our plans to test the local hypothesis for these high-velocity clouds (HVCs) by observing the interstellar Mg II absorption toward several stars in the SN1991T/3C273 field with IUE. Although the many HVCs observed in H I 21 cm emission have not yet been conclusively detected in metal absorption against any Galactic source (Wakker 1991), our particular search is worthwhile given that: (1) the general issue of HVC distances remains unclear due to metallicity uncertainties and, (2) the SN1991T/3C273 sightline presents a possibly unique case where there is radio continuum evidence of a local disturbance.

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

    PubMed

    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.

  8. 880 μm SMA Polarization Observations of the Quasar 3C 286

    NASA Astrophysics Data System (ADS)

    Hull, Charles L. H.; Girart, Josep M.; Zhang, Qizhou

    2016-10-01

    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° at λ ≳ 3 cm to ∼38° at λ ≈ 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 μm (340 GHz) with the Submillimeter Array. We find a polarization position angle and percentage of 37\\buildrel{\\circ}\\over{.} 4+/- 1\\buildrel{\\circ}\\over{.} 5 and 15.7% ± 0.8%, respectively, which is consistent with previous measurements at 1 mm.

  9. 40 CFR 59.1 - Final determinations under Section 183(e)(3)(C) of the CAA.

    Code of Federal Regulations, 2013 CFR

    2013-07-01

    ... 40 Protection of Environment 6 2013-07-01 2013-07-01 false Final determinations under Section 183(e)(3)(C) of the CAA. 59.1 Section 59.1 Protection of Environment ENVIRONMENTAL PROTECTION AGENCY (CONTINUED) AIR PROGRAMS (CONTINUED) NATIONAL VOLATILE ORGANIC COMPOUND EMISSION STANDARDS FOR CONSUMER...

  10. Tobacco Etch Virus protease: A shortcut across biotechnologies.

    PubMed

    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. PMID:27312702

  11. Tobacco Etch Virus protease: A shortcut across biotechnologies.

    PubMed

    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.

  12. Allostery in trypsin-like proteases suggests new therapeutic strategies.

    PubMed

    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.

  13. Alkaline protease from Neurospora crassa. Purification and partial characterization

    SciTech Connect

    Lindberg, R.A.; Eirich, L.D.; Price, J.S.; Wolfinbarger, L. Jr.; Drucker, H.

    1981-01-25

    A simple purification procedure was developed for the extracellular alkaline protease from Neurospora crassa. Key steps in the purification were: (1) the choice of gelatin as the protein inducer, which induces optimally at a much lower concentration than other commonly employed protein inducers; (2) heat treatment, during which the inducer is digested by the protease; and (3) a concentration step that eliminates the usual precipitation procedures and removes much of the digested protein inducer. The preparation was homogeneous and had a molecular weight of approx. 30,500. The protease has 100% activity from pH 6.0 to 10.0, is heat labile above 45/sup 0/C, and susceptible to autodigestion. Hydrolysis of the ..beta.. chain from insulin indicates a preferential cleavage on the carboxyl group side of neutral and aromatic amino acids.

  14. An Augmented Multiple-Protease-Based Human Phosphopeptide Atlas.

    PubMed

    Giansanti, Piero; Aye, Thin Thin; van den Toorn, Henk; Peng, Mao; van Breukelen, Bas; Heck, Albert J R

    2015-06-23

    Although mass-spectrometry-based screens enable thousands of protein phosphorylation sites to be monitored simultaneously, they often do not cover important regulatory sites. Here, we hypothesized that this is due to the fact that nearly all large-scale phosphoproteome studies are initiated by trypsin digestion. We tested this hypothesis using multiple proteases for protein digestion prior to Ti(4+)-IMAC-based enrichment. This approach increases the size of the detectable phosphoproteome substantially and confirms the considerable tryptic bias in public repositories. We define and make available a less biased human phosphopeptide atlas of 37,771 unique phosphopeptides, correlating to 18,430 unique phosphosites, of which fewer than 1/3 were identified in more than one protease data set. We demonstrate that each protein phosphorylation site can be linked to a preferred protease, enhancing its detection by mass spectrometry (MS). For specific sites, this approach increases their detectability by more than 1,000-fold. PMID:26074081

  15. Substrate specificity of the ubiquitin and Ubl proteases

    PubMed Central

    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

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

    PubMed

    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.

  17. Protease-triggered siRNA delivery vehicles.

    PubMed

    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.

  18. Membrane-anchored serine proteases in health and disease

    PubMed Central

    Bugge, Thomas; Wu, Qingyu

    2013-01-01

    Serine proteases of the trypsin-like family have long been recognized to be critical effectors of biological processes as diverse as digestion, blood coagulation, fibrinolysis, and immunity. In recent years, a subgroup of these enzymes has been identified that are anchored directly to plasma membranes, either by a carboxy-terminal transmembrane domain (Type I), an amino-terminal transmembrane domain with a cytoplasmic extension (Type II or TTSP), or through a glycosyl-phosphatidylinositol (GPI) linkage. Recent biochemical, cellular, and in vivo analyses have now established that membrane-anchored serine proteases are key pericellular contributors to processes vital for development and the maintenance of homeostasis. This chapter will review our current knowledge of the biological and physiological functions of these proteases, their molecular substrates, and their contributions to disease. PMID:21238933

  19. Acid phosphatase and protease activities in immobilized rat skeletal muscles

    NASA Technical Reports Server (NTRS)

    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.

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

    PubMed

    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

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

    SciTech Connect

    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.

  2. Structural evaluation of three 2-phenylpyrazolo[4,3-c]quinolin-3-one monohydrates

    NASA Astrophysics Data System (ADS)

    Ferreira, Vitor F.; Leal, Katia Z.; Lindgren, Eric B.; de Oliveira, Mara R. P.; de Souza, Maria Celia B. V.; Vasconcelos, Thatyana R. A.; Wardell, James L.; Wardell, Solange M. S. V.; Yoneda, Julliane D.

    2013-11-01

    A single crystal X-ray diffraction and theoretical study has been carried out on mono hydrates of three 2H-pyrazolo[4,3-c]quinolin-3(5H)-one derivatives, namely 6-methyl-2-phenylpyrazolo[4,3-c]quinolin-3-one, 3, 6-methyl-2-(4-chlorophenyl)pyrazolo[4,3-c]quinolin-3-one, 4, and 8-methyl-2-(4-nitrophenyl)pyrazolo[4,3-c]quinolin-3-one, 5. The monohydrates were obtained on recrystallization from moist solvents. While there are three tautomeric forms possible for such pyrazolo[4,3-c]quinolin-3-one molecules, the sole form isolated in the solid [(X)ṡ(H2O)] (X = 3, 4 and 5) compounds was the quinoloid form - the one calculated to be the most stable at the M06-2X/6-311++G(d,p) level of theory. Excellent agreement was found between the calculated and X-ray determined structures. Molecule 5 in [(5)ṡ(H2O)] is very near planar while both molecules 3 and 4 in their respective hydrates are much less so as a consequence of angles about 24° between the two aromatic rings. In each hydrate, the pyrazolo[4,3-c]quinolin-3-one molecule is bonded to three water molecules and each water molecule is likewise H-bonded to three pyrazolo[4,3-c]quinolin-3-one molecules. While the water molecules are H-bonded to 3 and 4 via the pyridinyl N and 2x the carbonyl O atoms, in [(5)ṡ(H2O)] the H-bonds are to pyridinyl N, carbonyl O and a nitro O atoms. Calculations indicated that the found arrangement in [(5)ṡ(H2O)] is more stable than one using the connections as found in [(3)ṡ(H2O)] and [(4)ṡ(H2O)]. While each of the hydrates possess strong Nsbnd H⋯O and Osbnd H⋯O hydrogen bonds, and weaker Csbnd H⋯π and π⋯π interactions, the supramolecular arrays are very different.

  3. Genetic Changes in HIV-1 Gag-Protease Associated with Protease Inhibitor-Based Therapy Failure in Pediatric Patients

    PubMed Central

    Giandhari, Jennifer; Basson, Adriaan E.; Coovadia, Ashraf; Kuhn, Louise; Abrams, Elaine J.; Strehlau, Renate; Morris, Lynn

    2015-01-01

    Abstract Studies have shown a low frequency of HIV-1 protease drug resistance mutations in patients failing protease inhibitor (PI)-based therapy. Recent studies have identified mutations in Gag as an alternate pathway for PI drug resistance in subtype B viruses. We therefore genotyped the Gag and protease genes from 20 HIV-1 subtype C-infected pediatric patients failing a PI-based regimen. Major protease resistance mutations (M46I, I54V, and V82A) were identified in eight (40%) patients, as well as Gag cleavage site (CS) mutations (at codons 373, 374, 378, 428, 431, 449, 451, and 453) in nine (45%) patients. Four of these Gag CS mutations occurred in the absence of major protease mutations at PI failure. In addition, amino acid changes were noted at Gag non-CS with some predicted to be under HLA/KIR immune-mediated pressure and/or drug selection pressure. Changes in Gag during PI failure therefore warrant further investigation of the Gag gene and its role in PI failure in HIV-1 subtype C infection. PMID:25919760

  4. Gag-Protease Sequence Evolution Following Protease Inhibitor Monotherapy Treatment Failure in HIV-1 Viruses Circulating in East Africa.

    PubMed

    Sutherland, Katherine A; Goodall, Ruth L; McCormick, Adele; Kapaata, Anne; Lyagoba, Fred; Kaleebu, Pontiano; Thiltgen, Geant; Gilks, Charles F; Spyer, Moira; Kityo, Cissy; Pillay, Deenan; Dunn, David; Gupta, Ravindra K

    2015-10-01

    Around 2.5 million HIV-infected individuals failing first-line therapy qualify for boosted protease inhibitor (bPI)-based second-line therapy globally. Major resistance mutations are rarely present at treatment failure in patients receiving bPI and the determinants of failure in these patients remain unknown. There is evidence that Gag can impact PI susceptibility. Here, we have sequenced Gag-Protease before and following failure in 23 patients in the SARA trial infected with subtypes A, C, and D viruses. Before bPI, significant variation in Protease and Gag was observed at positions previously associated with PI exposure and resistance including Gag mutations L449P, S451N, and L453P and Protease K20I and L63P. Following PI failure, previously described mutations in Protease and Gag were observed, including those at the cleavage sites such as R361K and P453L. However, the emergence of clear genetic determinants of therapy failure across patients was not observed. Larger Gag sequence datasets will be required to comprehensively identify mutational correlates of bPI failure across subtypes. PMID:26258548

  5. Thermal expansion of Fe3C at high pressure and carbon in the Earth's inner core

    NASA Astrophysics Data System (ADS)

    Gao, L.; Chen, B.; Wang, Y.; Li, J.

    2008-12-01

    Carbon is one of the major candidates for the principal light element in the Earth's core. Wood [1993] proposed that Fe3C, rather than iron-nickel alloy, is the dominant phase in the Earth's solid inner core. Testing the model of Fe3C-rich inner core requires knowledge on the thermal equation-of-state (EoS) of Fe3C under core conditions. To date, EoS data of Fe3C are only available at high pressure and 0 or 300 K [Scott et al., 2001, Li et al., 2002, Vocadlo et al., 2002] or at high temperature and 1 bar [Wood et al., 2004]. Wood et al. [2004] found that the thermal expansion coefficient is significantly affected by the ferromagnetic to paramagnetic transition above the Curie temperature. In this study, we have determined the thermal expansion coefficient of Fe3C up to 20 GPa and 1273 K, using a T-cup device and synchrotron x-ray diffraction techniques at beamline 13-ID of the Advanced Photon Source. Our results place constraints on the abundance of carbon the Earth's inner core. This work is supported by NSF EAR 06-09639. References: Gao et al. (2008), Geophys. Res. Lett., doi:10.1029/2008GL034817. Li, J. et al. (2002), Phys. Chem. Miner., 29(3), 166-169. Scott, H. P. et al. (2001), Geophys. Res. Lett., 28, 1875-1878 Vocadlo, L., et al. (2002), Earth Planet. Sci. Lett., 203(1), 567-575. 347. Wood, B. J. (1993), Earth Planet. Sci. Lett., 117(3-4), 593-607. Wood, I. G. et al. (2004), J. Appl. Crystallogr., 37, 82-90.

  6. Alkyl hydroxybenzoic acid derivatives that inhibit HIV-1 protease dimerization.

    PubMed

    Flausino, O A; Dufau, L; Regasini, L O; Petrônio, M S; Silva, D H S; Rose, T; Bolzani, V S; Reboud-Ravaux, M

    2012-01-01

    The therapeutic potential of gallic acid and its derivatives as anti-cancer, antimicrobial and antiviral agents is well known. We have examined the mechanism by which natural gallic acid and newly synthesized gallic acid alkyl esters and related protocatechuic acid alkyl esters inhibit HIV-1 protease to compare the influence of the aromatic ring substitutions on inhibition. We used Zhang-Poorman's kinetic analysis and fluorescent probe binding to demonstrate that several gallic and protecatechuic acid alkyl esters inhibited HIV-1 protease by preventing the dimerization of this obligate homodimeric aspartic protease rather than targeting the active site. The tri-hydroxy substituted benzoic moiety in gallates was more favorable than the di-substituted one in protocatechuates. In both series, the type of inhibition, its mechanism and the inhibitory efficiency dramatically depended on the length of the alkyl chain: no inhibition with alkyl chains less than 8 carbon atoms long. Molecular dynamics simulations corroborated the kinetic data and propose that gallic esters are intercalated between the two N- and C-monomer ends. They complete the β-sheet and disrupt the dimeric enzyme. The best gallic ester (14 carbon atoms, K(id) of 320 nM) also inhibited the multi-mutated protease MDR-HM. These results will aid the rational design of future generations of non-peptide inhibitors of HIV-1 protease dimerization that inhibit multi-mutated proteases. Finally, our work suggests the wide use of gallic and protocatechuic alkyl esters to dissociate intermolecular β-sheets involved in protein-protein interactions.

  7. Analysis of the immunoglobulin A protease gene of Streptococcus sanguis.

    PubMed Central

    Gilbert, J V; Plaut, A G; Wright, A

    1991-01-01

    The amino acid sequence T-P-P-T-P-S-P-S is tandemly duplicated in the heavy chain of human immunoglobulin A1 (IgA1), the major antibody in secretions. The bacterial pathogen Streptococcus sanguis, a precursor to dental caries and a cause of bacterial endocarditis, yields IgA protease that cleaves only the Pro-Thr peptide bond in the left duplication, while the type 2 IgA proteases of the genital pathogen Neisseria gonorrhoeae and the respiratory pathogen Haemophilus influenzae cleave only the P-T bond in the right half. We have sequenced the entire S. sanguis iga gene cloned into Escherichia coli. A segment consisting of 20 amino acids tandemly repeated 10 times, of unknown function, occurs near the amino-terminal end of the enzyme encoded in E. coli. Identification of a predicted zinc-binding region in the S. sanguis enzyme and the demonstration that mutations in this region result in production of a catalytically inactive protein support the idea that the enzyme is a metalloprotease. The N. gonorrhoeae and H. influenzae enzymes were earlier shown to be serine-type proteases, while the Bacteroides melaninogenicus IgA protease was shown to be a cysteine-type enzyme. The streptococcal IgA protease amino acid sequence has no significant homology with either of the two previously determined IgA protease sequences, that of type 2 N. gonorrhoeae and type 1 H. influenzae. The differences in both structure and mechanism among these functionally analogous enzymes underscore their role in the infectious process and offer some prospect of therapeutic intervention. Images PMID:1987065

  8. [Isolation of Actinomycetes synthesizing proteases with thrombolytic activity].

    PubMed

    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

  9. The chlamydial protease CPAF: important or not, important for what?

    PubMed

    Häcker, Georg

    2014-05-01

    The protease CPAF is only found in Chlamydiales and in at least most bacteria that share with Chlamydia the biphasic life-style in a cytosolic inclusion. CPAF is intriguing: it appears to be secreted from the inclusion across the inclusion membrane into the cytosol. A bacterial protease ravaging in the cytosol of a human cell may cause a plethora of effects. Curiously, very few are known. The current discussion is bogged down by a focus on experimental artifact, while proposed functions of CPAF remain speculative. I here make the attempt to summarize what we know about CPAF.

  10. Rhomboid proteases in mitochondria and plastids: keeping organelles in shape.

    PubMed

    Jeyaraju, Danny V; Sood, Aditi; Laforce-Lavoie, Audrey; Pellegrini, Luca

    2013-02-01

    Rhomboids constitute the most widespread and conserved family of intramembrane cleaving proteases. They are key regulators of critical cellular processes in bacteria and animals, and are poised to play an equally important role also in plants. Among eukaryotes, a distinct subfamily of rhomboids, prototyped by the mammalian mitochondrial protein Parl, ensures the maintenance of the structural and functional integrity of mitochondria and plastids. Here, we discuss the studies that in the past decade have unveiled the role, regulation, and structure of this unique group of rhomboid proteases. This article is part of a Special Issue entitled: Protein Import and Quality Control in Mitochondria and Plastids.

  11. A CHANDRA SNAPSHOT SURVEY FOR 3C RADIO GALAXIES WITH REDSHIFTS BETWEEN 0.3 AND 0.5

    SciTech Connect

    Massaro, F.; Harris, D. E.; Paggi, A.; Tremblay, G. R.; Liuzzo, E.; Bonafede, A.

    2013-05-01

    This paper contains an analysis of short Chandra observations of 19 3C sources with redshifts between 0.3 and 0.5 not previously observed in the X-rays. This sample is part of a project to obtain Chandra data for all of the extragalactic sources in the 3C catalog. Nuclear X-ray intensities as well as any X-ray emission associated with radio jet knots, hotspots, or lobes have been measured in three energy bands: soft, medium, and hard. Standard X-ray spectral analysis for the four brightest nuclei has also been performed. X-ray emission was detected for all the nuclei of the radio sources in the current sample with the exception of 3C 435A. There is one compact steep spectrum source while all the others are FR II radio galaxies. X-ray emission from two galaxy clusters (3C 19 and 3C 320), from six hotspots in four radio galaxies (3C 16, 3C 19, 3C 268.2, 3C 313), and extended X-ray emission on kiloparsec scales in 3C 187 and 3C 313, has been detected.

  12. Semaphorin 3C Released from a Biocompatible Hydrogel Guides and Promotes Axonal Growth of Rodent and Human Dopaminergic Neurons.

    PubMed

    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

  13. Semaphorin 3C Released from a Biocompatible Hydrogel Guides and Promotes Axonal Growth of Rodent and Human Dopaminergic Neurons

    PubMed Central

    Carballo-Molina, Oscar A.; Sánchez-Navarro, Andrea; López-Ornelas, Adolfo; Lara-Rodarte, Rolando; Salazar, Patricia; Campos-Romo, Aurelio; Ramos-Mejía, Verónica

    2016-01-01

    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

  14. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases.

    PubMed

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex with anti-proteases, at higher concentrations. Data from pharmacokinetic investigations reveals dose linearity for maximum plasma levels of free proteases not unusual for body proteases and a high inter-individual variability. There is no interference with each other after oral administration of protease combinations, and absorption follows an unusual invasion and elimination kinetic due to slow velocity of absorption and a fast 100% protein binding to anti-proteases. Oral application of proteases leads to increased proteolytic serum activity and increased plasma concentrations of the corresponding anti-proteases. Their biological activity is determined by their proteolytic activity as free proteases on soluble peptides/proteins or cell surface receptors (e.g. protease activated receptors) and their activity in the complex formed with their specific and/or unspecific anti-proteases. The anti-protease-complexes, during immune reaction and injuries often loaded with different cytokines, are cleared from body fluids and tissue by receptor mediated endocytosis on hepatocytes and/or blood cells. Oral administration of enteric coated tablets containing proteolytic enzymes of plant and animal origin may be a safe method to stabilize, positively influence or enhance physiological and immunological processes during disease processes and in healthy consumers.

  15. Gastrointestinal absorption and biological activities of serine and cysteine proteases of animal and plant origin: review on absorption of serine and cysteine proteases

    PubMed Central

    Lorkowski, Gerhard

    2012-01-01

    Research has confirmed that peptides and larger protein molecules pass through the mucosal barrier of the gastrointestinal tract. Orally administered serine and cysteine proteases of plant and animal origin also reach blood and lymph as intact, high molecular weight and physiologically active protein molecules. Their absorption may be supported by a self-enhanced paracellular transport mechanism resulting in sub-nanomolar concentration of transiently free protease molecules or, in a complex with anti-proteases, at higher concentrations. Data from pharmacokinetic investigations reveals dose linearity for maximum plasma levels of free proteases not unusual for body proteases and a high inter-individual variability. There is no interference with each other after oral administration of protease combinations, and absorption follows an unusual invasion and elimination kinetic due to slow velocity of absorption and a fast 100% protein binding to anti-proteases. Oral application of proteases leads to increased proteolytic serum activity and increased plasma concentrations of the corresponding anti-proteases. Their biological activity is determined by their proteolytic activity as free proteases on soluble peptides/proteins or cell surface receptors (e.g. protease activated receptors) and their activity in the complex formed with their specific and/or unspecific anti-proteases. The anti-protease-complexes, during immune reaction and injuries often loaded with different cytokines, are cleared from body fluids and tissue by receptor mediated endocytosis on hepatocytes and/or blood cells. Oral administration of enteric coated tablets containing proteolytic enzymes of plant and animal origin may be a safe method to stabilize, positively influence or enhance physiological and immunological processes during disease processes and in healthy consumers. PMID:22461953

  16. Genetic Evidence Supporting the Association of Protease and Protease Inhibitor Genes with Inflammatory Bowel Disease: A Systematic Review

    PubMed Central

    Bekkering, Geertruida E.; Nüesch, Eveline; Mendes, Camila T.; Schmied, Stefanie; Wyder, Stefan; Kellen, Eliane; Villiger, Peter M.; Rutgeerts, Paul; Vermeire, Séverine; Lottaz, Daniel

    2011-01-01

    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 4protease 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. PMID:21931648

  17. A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles.

    PubMed

    Gullberg, Maria; Lohse, Louise; Bøtner, Anette; McInerney, Gerald M; Burman, Alison; Jackson, Terry; Polacek, Charlotta; Belsham, Graham J

    2016-01-01

    Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvβ6 (a cellular receptor for FMDV). In cattle vaccinated with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge, they were protected against disease and no FMDV RNA was detected in their sera post-challenge. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed. This prime-boost system, using reagents that can be generated outside of high-containment facilities

  18. A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a “Single-Cycle” Alphavirus Vector and Empty Capsid Particles

    PubMed Central

    Gullberg, Maria; Lohse, Louise; Bøtner, Anette; McInerney, Gerald M.; Burman, Alison; Jackson, Terry; Polacek, Charlotta

    2016-01-01

    Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a “single cycle” packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvβ6 (a cellular receptor for FMDV). In cattle vaccinated with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge, they were protected against disease and no FMDV RNA was detected in their sera post-challenge. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed. This prime-boost system, using reagents that can be generated outside of high

  19. A Prime-Boost Vaccination Strategy in Cattle to Prevent Foot-and-Mouth Disease Using a "Single-Cycle" Alphavirus Vector and Empty Capsid Particles.

    PubMed

    Gullberg, Maria; Lohse, Louise; Bøtner, Anette; McInerney, Gerald M; Burman, Alison; Jackson, Terry; Polacek, Charlotta; Belsham, Graham J

    2016-01-01

    Foot-and-mouth disease (FMD) remains one of the most economically important infectious diseases of production animals globally. Vaccination can successfully control this disease, however, current vaccines are imperfect. They are made using chemically inactivated FMD virus (FMDV) that is produced in large-scale mammalian cell culture under high containment conditions. Here, we have expressed the FMDV capsid protein precursor (P1-2A) of strain O1 Manisa alone or with the FMDV 3C protease (3Cpro) using a "single cycle" packaged alphavirus self-replicating RNA based on Semliki Forest virus (SFV). When the FMDV P1-2A was expressed with 3Cpro then processing of the FMDV capsid precursor protein is observed within cells and the proteins assemble into empty capsid particles. The products interact with anti-FMDV antibodies in an ELISA and bind to the integrin αvβ6 (a cellular receptor for FMDV). In cattle vaccinated with these rSFV-FMDV vectors alone, anti-FMDV antibodies were elicited but the immune response was insufficient to give protection against FMDV challenge. However, the prior vaccination with these vectors resulted in a much stronger immune response against FMDV post-challenge and the viremia observed was decreased in level and duration. In subsequent experiments, cattle were sequentially vaccinated with a rSFV-FMDV followed by recombinant FMDV empty capsid particles, or vice versa, prior to challenge. Animals given a primary vaccination with the rSFV-FMDV vector and then boosted with FMDV empty capsids showed a strong anti-FMDV antibody response prior to challenge, they were protected against disease and no FMDV RNA was detected in their sera post-challenge. Initial inoculation with empty capsids followed by the rSFV-FMDV was much less effective at combating the FMDV challenge and a large post-challenge boost to the level of anti-FMDV antibodies was observed. This prime-boost system, using reagents that can be generated outside of high-containment facilities

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

    ERIC Educational Resources Information Center

    Johnson, R. Jeremy

    2014-01-01

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

  1. A New Subtilase-Like Protease Deriving from Fusarium equiseti with High Potential for Industrial Applications.

    PubMed

    Juntunen, Kari; Mäkinen, Susanna; Isoniemi, Sari; Valtakari, Leena; Pelzer, Alexander; Jänis, Janne; Paloheimo, Marja

    2015-09-01

    A gene encoding a novel extracellular subtilisin-like protease was cloned from the ascomycete Fusarium equiseti and expressed in Trichoderma reesei. The F. equiseti protease (Fe protease) showed excellent performance in stain removal and good compatibility with several commercial laundry detergent formulations, suggesting that it has high potential for use in various industrial applications. The recombinant enzyme was purified and characterized. The temperature optimum of the Fe protease was 60 °C and it showed high activity in the pH range of 6-10, with a sharp decline in activity at pH above 10. The amino acid specificity of the Fe protease was studied using casein, cytochrome c, and ubiquitin as substrates. The Fe protease had broad substrate specificity: almost all amino acid residues were accepted at position P1, even though it showed some preference for cleavage at the C-terminal side of asparagine and histidine residues. The S4 subsite of Fe protease favors aspartic acid and threonine. The other well-characterized proteases from filamentous fungi, Proteinase K from Engyodontium album, Thermomycolin from Malbranchea sulfurea, and alkaline subtilisins from Bacillus species prefer hydrophobic amino acids in both the S1 and S4 subsites. Due to its different specificity compared to the members of the S8 family of clan SB of proteases, we consider that the Fe protease is a new protease. It does not belong to any previously defined IUBMB groups of proteases.

  2. The synthesis of size-controlled 3C-SiC nanoflakes and their photoluminescent properties

    NASA Astrophysics Data System (ADS)

    Fan, Xiujun; Ye, Ruquan; Peng, Zhiwei; Wang, Juanjuan; Fan, Ailing; Guo, Xia

    2016-06-01

    Size-controlled and high-purity 3C-SiC nanoflakes (NFs) are synthesized on the tips of vertically aligned carbon nanotube (VA-CNT) carpets with a hot-filament chemical vapor deposition (HF-CVD) method. The average diameter and height of SiC NFs can be tuned by changing the thickness of per-deposited Si and growth conditions. The growth process of the SiC NFs is suggested to be dominated by a vapor-solid (VS) mechanism. The prepared SiC NFs exhibit quantum-confinement effects, emitting strong violet-blue photoluminescence (PL) under ultraviolet excitation. The PL peak position changes from 410 to 416 nm as the excitation line increases from 290 to 400 nm. This result opens the possibility for the application of the luminescent solid-state freestanding 3C-SiC NFs in photonics as well as photonics/electronics integration.

  3. Time constant of defect relaxation in ion-irradiated 3C-SiC

    SciTech Connect

    Wallace, J. B.; Bayu Aji, L. B.; Kucheyev, S. O.; Shao, L.

    2015-05-18

    Above room temperature, the buildup of radiation damage in SiC is a dynamic process governed by the mobility and interaction of ballistically generated point defects. Here, we study the dynamics of radiation defects in 3C-SiC bombarded at 100 °C with 500 keV Ar ions, with the total ion dose split into a train of equal pulses. Damage–depth profiles are measured by ion channeling for a series of samples irradiated under identical conditions except for different durations of the passive part of the beam cycle. Results reveal an effective defect relaxation time constant of ∼3 ms (for second order kinetics) and a dynamic annealing efficiency of ∼40% for defects in both Si and C sublattices. This demonstrates a crucial role of dynamic annealing at elevated temperatures and provides evidence of the strong coupling of defect accumulation processes in the two sublattices of 3C-SiC.

  4. Optical and Hubble Space Telescope ultraviolet spectropolarimetry of 3C 273 and PG 1114+445

    NASA Technical Reports Server (NTRS)

    Smith, Paul S.; Schmidt, Gary D.; Allen, Richard G.

    1993-01-01

    New optical spectropolarimetry and broad-band measurements, made during periods when the linear polarization of the quasar 3C 273 was no greater than 1 pct, are combined with ultraviolet polarization measurements made with the Faint Object Spectrograph of the Hubble Space Telescope. Very weak UV polarization during the time of observation is seen in the HST measurements, which is consistent with the completely unpolarized UV continuum and emission lines. Optical spectropolarimetry, however, reveals polarized light, the source of which is consistent with a nonthermal component, diluted by other unpolarized emission sources, such as the 'big blue bump'. Optical spectropolarimetry of the radio-quiet quasar PG 1114+445 is also presented. The polarization of this QSO is thought to result from scattering by dust external to the narrow-line region. 3C 273 and PG 1114+445 are useful in illustrating the range of polarizing mechanisms which can operate in low-polarization QSOs.

  5. Origin of X-rays and nature of accretion in the radio galaxy 3C 88

    NASA Astrophysics Data System (ADS)

    Gliozzi, Mario

    2005-10-01

    Studies of stellar dynamics have established that the presence of supermassive black holes is almost ubiquitous not only in AGN, but also in normal galaxies. Therefore, of crucial importance is the role played by low-power AGN, which represent the link between powerful AGN and normal galaxies. We propose to observe (35 ks) the radio galaxy 3C88, which complements and extends towards lower X-ray luminosities our sample of low-power radio galaxies hosting a LINER. 3C88 is an FRII/LINER which hosts a black hole of estimated mass. Specific goals are: investigate the origin of X-rays (jet vs. accretion related emission); assess the nature of the accretion in low-power objects; investigate possible intrinsic differences in the X-ray properties of FRIs and FRIIs.

  6. Spin-glass transition in Ni carbide single crystal nanoparticles with Ni3C - type structure

    NASA Astrophysics Data System (ADS)

    Fujieda, S.; Kuboniwa, T.; Shinoda, K.; Suzuki, S.; Echigoya, J.

    2016-05-01

    Hexagonal shaped nanoparticles about 60 nm in size were successfully synthesized in tetraethylene glycol solution containing polyvinylpyrrolidone. By the analysis of the electron diffraction pattern, these were identified as a single crystal of Ni carbide with Ni3C - type structure. Their magnetization curve at 5 K was not completely saturated under a magnetic field of 5 T. The thermomagnetization curves after zero-field cooling and after field cooling exhibited the magnetic cooling effect at low temperatures. Furthermore, the 2nd order nonlinear term of AC magnetic susceptibility exhibited a negative divergence at about 17 K. It is concluded that Ni carbide single crystal nanoparticles with the Ni3C - type structure exhibit spin-glass transition at low temperatures.

  7. The picornaviral 3C proteinases: cysteine nucleophiles in serine proteinase folds.

    PubMed Central

    Malcolm, B. A.

    1995-01-01

    The 3C proteinases are a novel group of cysteine proteinases with a serine proteinase-like fold that are responsible for the bulk of polyprotein processing in the Picornaviridae. Because members of this viral family are to blame for several ongoing global pandemic problems (rhinovirus, hepatitis A virus) as well as sporadic outbreaks of more serious pathologies (poliovirus), there has been continuing interest over the last two decades in the development of antiviral therapies. The recent determination of the structure of two of the 3C proteinases by X-ray crystallography opens the door for the application of the latest advances in computer-assisted identification and design of anti-proteinase therapeutic/chemoprophylactic agents. PMID:8520469

  8. PTOSL response of commercial Al2O3:C detectors to ultraviolet radiation.

    PubMed

    Gronchi, Claudia C; Caldas, Linda V E

    2013-04-01

    The photo-transferred optically stimulated luminescence (PTOSL) technique using Al2O3:C detectors has been suggested as a good option for ultraviolet (UV) radiation dosimetry. The objective of this work was to study the PTOSL response of Al2O3:C InLight detectors and the OSL microStar reader of Landauer. The parameters such as radiation pre-dose, optical treatment time and UV illumination time were determined. The detectors presented a satisfactory stimulus of PTOSL signals when they were subjected to a preconditioning procedure with gamma radiation (1 Gy pre-dose), 30 min of optical treatment (to empty the shallow traps) and 30 min of UV illumination from an artificial source. PMID:22887115

  9. Environmental variability and the evolution of the glucocorticoid receptor (Nr3c1) in African starlings.

    PubMed

    Hofmeister, Natalie R; Rubenstein, Dustin R

    2016-10-01

    One of the primary ways that organisms cope with environmental change is through regulation of the hypothalamo-pituitary-adrenal (HPA) axis, the neuroendocrine system that controls reactions to stress. Variation in genes regulating the HPA axis - particularly the glucocorticoid receptor - may facilitate adaptation to changing climatic conditions by altering expression. Here we examine signatures of selection on the glucocorticoid receptor gene (Nr3c1) in African starlings that inhabit a range of environments, including those with variable climatic conditions. To investigate potential adaptive mechanisms underlying the vertebrate stress response, we sequence the Nr3c1 gene in 27 species of African starlings. Although we find some evidence of positive selection, substitution rate is negatively correlated with variance in precipitation. This suggests climatic cycling in sub-Saharan Africa may have resulted in lower substitution rates to maintain a successful coping strategy. When environmental conditions fluctuate rapidly, variation in the strength of purifying selection can explain evolutionary rate variation. PMID:27500971

  10. COMPTEL gamma-ray observations of the quasars CTA 102 and 3C 454.3

    NASA Technical Reports Server (NTRS)

    Blom, J. J.; Bloemen, H.; Bennett, K.; Collmar, W.; Hermsen, W.; Mcconnell, M.; Schoenfelder, V.; Stacy, J. G.; Steinle, H.; Strong, A.

    1994-01-01

    The blazar-type active galactic nuclei CTA 102 (QSO 2230+114) and 3C 454.3 (QSO 2251+158), located about 7 deg apart, were observed by the Compton Gamma Ray Observatory at four epochs in 1992. Both were detected by Energy Gamma Ray Experiment Telescope (EGRET). The combined Compton Telescope (COMPTEL) observations in the 10-30 MeV energy range clearly indicate a source of MeV emission, which is likely due to a contribution from both quasars. These observations strongly suggest that the power-law spectra measured by EGRET above approximately 50 MeV flatten at lower MeV energies. A comparison with observations at other wavelengths shows that the power spectra of CTA 102 and 3C 454.3 peak at MeV energies. This behavior appears to be a common feature of gamma-ray active galactic nuclei (AGN).

  11. Fermi large area telescope observations of blazar 3C 279 occultations by the sun

    SciTech Connect

    Barbiellini, G.; Bastieri, D.; Buson, S.; Bechtol, K.; Blandford, R. D.; Borgland, A. W.; Buehler, R.; Cameron, R. A.; Chiang, J.; Bellazzini, R.; Bregeon, J.; Bruel, P.; Caraveo, P. A.; Cavazzuti, E.; Ciprini, S.; Cecchi, C.; Chaves, R. C. G.; Cheung, C. C. E-mail: phdmitry@stanford.edu; and others

    2014-04-01

    Observations of occultations of bright γ-ray sources by the Sun may reveal predicted pair halos around blazars and/or new physics, such as, e.g., hypothetical light dark matter particles—axions. We use Fermi Gamma-Ray Space Telescope (Fermi) data to analyze four occultations of blazar 3C 279 by the Sun on October 8 each year from 2008 to 2011. A combined analysis of the observations of these occultations allows a point-like source at the position of 3C 279 to be detected with significance of ≈3σ, but does not reveal any significant excess over the flux expected from the quiescent Sun. The likelihood ratio test rules out complete transparency of the Sun to the blazar γ-ray emission at a 3σ confidence level.

  12. OPTICAL MONITORING OF TWO BRIGHTEST NEARBY QUASARS, PHL 1811 AND 3C 273

    SciTech Connect

    Fan, J. H.; Liu, Y.; Yuan, Y. H.; Kurtanidze, O.; Chanishvili, R.; Richter, G. M.

    2014-08-01

    Variability is one of the most observable characteristics of active galactic nuclei, and it is important when considering the emission mechanism. In this paper, we report optical photometry monitoring of two nearby brightest quasars, PHL 1811 and 3C 273, using the ST-6 camera attached to the Newtonian focus and the Ap6E CCD camera attached to the primary focus of the 70 cm meniscus telescope at the Abastumani Observatory, Georgia. PHL 1811 was monitored during the period from 2002 September to 2012 December, while 3C 273 was monitored during the period from 1998 February to 2008 May. During our monitoring period, the two sources did not show any significant intra-day variability. The largest detected variations are ΔR = 0.112 ± 0.010 mag. for PHL 1811, ΔB = 0.595 ± 0.099 mag, ΔV = 0.369 ± 0.028 mag, ΔR = 0.495 ± 0.076 mag, and ΔI = 0.355 ± 0.009 mag for 3C 273. When the periodicity analysis methods are adopted for the observations of the sources, a period of p = 5.80 ± 1.12 yr is obtained for PHL 1811 in the R light curve in the present work, and periods of p = 21.10 ± 0.14, 10.00 ± 0.14, 7.30 ± 0.09, 13.20 ± 0.09, 2.10 ± 0.06, and 0.68 ± 0.05 yr are obtained for 3C 273 based on the data in the present work combined with historical works.

  13. 3. 'C.P. Reconstruction Rocklin to Colfax, Standard Double Track Tunnel ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    3. 'C.P. Reconstruction Rocklin to Colfax, Standard Double Track Tunnel Portal Stones, Wings Parallel to Center Line, Ring Stones,' Southern Pacific Standard Double-Track Tunnel, ca. 1909. Compare to photos in documentation sets for Tunnel 18 (HAER No. CA-197), Tunnel 34 (HAER No. CA-206), and Tunnel 1 (HAER No. CA-207). - Central Pacific Transcontinental Railroad, Sacramento to Nevada state line, Sacramento, Sacramento County, CA

  14. VizieR Online Data Catalog: VRIJHK photometry of 3C 279 (Sandrinelli+, 2016)

    NASA Astrophysics Data System (ADS)

    Sandrinelli, A.; Covino, S.; Dotti, M.; Treves, A.

    2016-07-01

    The starting point of the present investigation is the VRIJHK photometric observations obtained with the robotic Rapid Eye Mounting telescope (REM) at La Silla, which are described in detail in Sandrinelli et al. 2014 (cat. J/A+A/562/A79). We add to the data available in the above mentioned paper the REM photometry of 3C 279 (see Table2), which is unpublished thus far. (2 data files).

  15. Chandra Observations of the Nuclei of Radio Galaxies: 3C 295 and Hydra A

    NASA Technical Reports Server (NTRS)

    Harris, D. E.; McNamara, B. R.; David, L. P.; Lavoie, Anthony R. (Technical Monitor)

    2000-01-01

    The angular resolution available with Chandra allows us to isolate the X-ray emission from the nucleus of many radio galaxies and obtain their spectra. As expected from unification schemes, spectra so far obtained can best be interpreted as heavily absorbed power laws. We present the spectral parameters so derived for 3C 295 and Hydra A and compare them to data obtained at other wavelengths.

  16. Photographic photometry of compact extragalactic objects Optical variability of the quasar 3C 345

    NASA Astrophysics Data System (ADS)

    Babadzhanyants, M. K.; Belokon, E. T.; Denisenko, N. S.; Semenova, E. V.

    1985-08-01

    The results of 11-years photographic observations of optical variability of the quasar 3C 345 are presented. A flare with a timescale of the order of one year, similar to those of 1967 and 1971, was observed in 1982. From a comparison of the obtained series of observations with those of the Rosemary Hill Observatory, a conclusion is drawn on the absence of systematic variability at a timescale of 5 - 20 hours exceeding 0m.2 in magnitude.

  17. Photographic photometry of compact extragalactic objects Optical variability of the quasar 3C 345

    SciTech Connect

    Babadzhaniants, M.K.; Belokon, E.T.; Denisenko, N.S.; Semenova, E.V.

    1985-08-01

    An 11-yr (1973-1983) program of photographic observations at the Leningrad Byurakan station is reported, tracing the optical variability of the quasar 3C 345. A roughly 1-yr flare resembling those of 1967 and 1971 occurred in 1982. Comparison with the concurrent observations at Rosemary Hill, Florida, shows no appreciable systematic B-band fluctuations on time scales of 5-20 h. 10 references.

  18. NMR Study of the Superconducting Gap Variation near the Mott Transition in Cs3C60

    NASA Astrophysics Data System (ADS)

    Wzietek, P.; Mito, T.; Alloul, H.; Pontiroli, D.; Aramini, M.; Riccò, M.

    2014-02-01

    Former extensive studies of superconductivity in the A3C60 compounds, where A is an alkali metal, have led one to consider that Bardeen-Cooper-Schrieffer electron-phonon pairing prevails in those compounds, though the incidence of electronic Coulomb repulsion has been highly debated. The discovery of two isomeric fulleride compounds Cs3C60 which exhibit a transition with pressure from a Mott insulator (MI) to a superconducting (SC) state clearly reopens that question. Using pressure (p) as a single control parameter of the C60 balls lattice spacing, one can now study the progressive evolution of the SC properties when the electronic correlations are increased towards the critical pressure pc of the Mott transition. We have used C13 and Cs133 NMR measurements on the cubic phase A15-Cs3C60 just above pc=5.0(3) kbar, where the SC transition temperature Tc displays a dome shape with decreasing cell volume. From the T dependence below Tc of the nuclear spin lattice relaxation rate (T1)-1 we determine the electronic excitations in the SC state, that is 2Δ, the gap value. The latter is found to be largely enhanced with respect to the Bardeen-Cooper-Schrieffer value established in the case of dense A3C60 compounds. It even increases slightly with decreasing p towards pc, where Tc decreases on the SC dome, so that 2Δ /kBTc increases regularly upon approaching the Mott transition. These results bring clear evidence that the increasing correlations near the Mott transition are not significantly detrimental to superconductivity. They rather suggest that repulsive electron interactions might even reinforce elecron-phonon superconductivity, being then partly responsible for the large Tc values, as proposed by theoretical models taking the electronic correlations as a key ingredient.

  19. Discovery and optimization of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1.

    PubMed

    Hornberger, Keith R; Berger, Dan M; Crew, Andrew P; Dong, Hanqing; Kleinberg, Andrew; Li, An-Hu; Medeiros, Matthew R; Mulvihill, Mark J; Siu, Kam; Tarrant, James; Wang, Jing; Weng, Felix; Wilde, Victoria L; Albertella, Mark; Bittner, Mark; Cooke, Andrew; Gray, Michael J; Maresca, Paul; May, Earl; Meyn, Peter; Peick, William; Romashko, Darlene; Tanowitz, Michael; Tokar, Brianna

    2013-08-15

    The discovery and potency optimization of a series of 7-aminofuro[2,3-c]pyridine inhibitors of TAK1 is described. Micromolar hits taken from high-throughput screening were optimized for biochemical and cellular mechanistic potency to ~10nM, as exemplified by compound 12az. Application of structure-based drug design aided by co-crystal structures of TAK1 with inhibitors significantly shortened the number of iterations required for the optimization. PMID:23850198

  20. Optical Monitoring of Two Brightest Nearby Quasars, PHL 1811 and 3C 273

    NASA Astrophysics Data System (ADS)

    Fan, J. H.; Kurtanidze, O.; Liu, Y.; Richter, G. M.; Chanishvili, R.; Yuan, Y. H.

    2014-08-01

    Variability is one of the most observable characteristics of active galactic nuclei, and it is important when considering the emission mechanism. In this paper, we report optical photometry monitoring of two nearby brightest quasars, PHL 1811 and 3C 273, using the ST-6 camera attached to the Newtonian focus and the Ap6E CCD camera attached to the primary focus of the 70 cm meniscus telescope at the Abastumani Observatory, Georgia. PHL 1811 was monitored during the period from 2002 September to 2012 December, while 3C 273 was monitored during the period from 1998 February to 2008 May. During our monitoring period, the two sources did not show any significant intra-day variability. The largest detected variations are ΔR = 0.112 ± 0.010 mag. for PHL 1811, ΔB = 0.595 ± 0.099 mag, ΔV = 0.369 ± 0.028 mag, ΔR = 0.495 ± 0.076 mag, and ΔI = 0.355 ± 0.009 mag for 3C 273. When the periodicity analysis methods are adopted for the observations of the sources, a period of p = 5.80 ± 1.12 yr is obtained for PHL 1811 in the R light curve in the present work, and periods of p = 21.10 ± 0.14, 10.00 ± 0.14, 7.30 ± 0.09, 13.20 ± 0.09, 2.10 ± 0.06, and 0.68 ± 0.05 yr are obtained for 3C 273 based on the data in the present work combined with historical works.

  1. Fermi LAT detection of renewed and strong GeV activity from blazar 3C 279

    NASA Astrophysics Data System (ADS)

    Cutini, Sara

    2015-06-01

    The Large Area Telescope (LAT), one of two instruments on the Fermi Gamma-ray Space Telescope, has observed an intense gamma-ray flare from a source positionally consistent with the flat spectrum radio quasar 3C 279, also known as 3FGL J1256.1-0547 (Acero et al. 2015, APJS, 218, 23), with radio coordinates R.A.: 194.0465271 deg, Dec: -5.7893119 deg (J2000.0; Johnston et al. 1995, AJ, 110, 880).

  2. FY-3C/VIRR SST algorithm and cal/val activities at NSMC/CMA

    NASA Astrophysics Data System (ADS)

    Wang, Sujuan; Cui, Peng; Zhang, Peng; Ran, Maonong; Lu, Feng; Wang, Weihe

    2014-11-01

    The National Satellite Meteorological Center (NSMC)/CMA global sea surface temperature (SST) data are derived from measurements made by the Visible and Infrared Radiometer (VIRR) on board the FY-3 series polar orbiting satellites. Quality controlled in situ data from iQUAM (STAR/NESDIS/NOAA) is used in FY-3B/C VIRR matching procedure. The monthly matchup database (MDB) is created from FY-3C VIRR measurements paired with coincident SST measurements from buoys since November 2013. The satellite sensor's brightness temperature and buoy SST pairs are included in the MDB if they are coincident within 3km in space and 1 hour in time. Least-Square Regression is used for estimating the first-guess coefficient and SST residuals. Outliers are removed using Median±2STD, and the final coefficients of robust regression are estimated. A set of SST regression formalisms are tested base on NOAA- 19/AVHRR 2010 MDB. The test shows that, for daytime split-window nonlinear SST (NLSST) is the best, for nighttime triple-window MCSST (TCSST) is the best, which is agree with STAR/NESDIS's. The same regression analysis method also used on FY-3C/VIRR MDB. Compare with the three daytime SST algorithms and five nighttime SST algorithms, the best algorithm to retrieve FY-3C/VIRR SST for daytime is NLSST and for nighttime is TCSST. Compare with the coefficients of nighttime algorithm TCSST, it shows that for FY-3B/C VIRR SST, the contribution of 3.7μm band is smaller than split-window bands. The performance of 3.7μm band of FY-3C/VIRR is better than FY-3B/VIRR, but worse than NOAA-19/AVHRR.

  3. Resolution of the southern radio lobe of 3C 33 at 660 nm

    NASA Technical Reports Server (NTRS)

    Crane, Phillipe; Stiavelli, Massimo

    1992-01-01

    A strand of emission coincident with the peak in the radio emission at 2 and 6 cm was revealed by an HST planetary camera image of the southern radio lobe of 3C 33. The detailed morphological similarity between the optical and radio data conclusively confirms the common origin of the radio and optical emission. The question of whether or not the detailed correlations in emission seen in the jets can also be observed on the larger scales of radio lobes is addressed.

  4. Protease production by immobilized mycelia of Streptomyces fradiae

    SciTech Connect

    Kokubu, T.; Karube, I.; Suzuki, S.

    1981-01-01

    Streptomyces fradiae was immobilized in polyacrylamide gel prepared from 5% total acrylamide (90% acrylamide and 10% N,N-methylenebisacrylamide). Production of protease by the immobilized mycelia was attempted in a batch system. A dilute medium containing 0.5% starch, 0.5% meat extract, and 0.5% yeast extract was employed. The reusability of the immobilized and washed mycelia was examined. The activity of protease production by washed mycelia was rapidly decreased with increasing use cycles. The activity of the immobilized mycelia increased gradually, and reached a maximum after ten use cycles. Then, the activity gradually decreased with increasing reaction cycles. This might be caused by destruction of the gels. On the other hand, the sterilization of the surface of the immobilized mycelia was effective for elongation of the lifetime. As a result, the half-life of protease production by the sterilized immobilized mycelia was about 30 days. The rate of protease production by immobilized mycelia was 12,000 U/ml/hr. This value was four times higher than that by submerged culture.

  5. Purification and characterization of a pineapple crown leaf thiol protease.

    PubMed

    Singh, L Rupachandra; Devi, Th Premila; Devi, S Kunjeshwori

    2004-02-01

    A thiol protease was isolated and purified from the crown leaf of pineapple, Ananas comosus (L.) Merr. cv. Queen, by an immunoaffinity procedure. After the purification to electrophoretic homogeneity, the enzyme was characterized with respect to some of its physico-chemical and kinetic properties. The molecular weight of the protease (22.4-22.9 kDa), Km (97 microM) and kcat (8.8 s(-1)) for its esterolytic cleavage of the synthetic protease substrate N(alpha)-CBZ-L-lysine p-nitrophenyl ester, the concentration of its thiol activator L-cysteine required for half maximal activation A0.5 (9.9 microM), optimum pH (6.5) for its proteolytic action on azocasein, T(1/2) (60 degrees C) for inactivation by heating the enzyme (35.5 microg protein/mL) in citrate buffer pH 6.0 for 15 min, and SH-group content (0.98 mol/mol enzyme) were determined. Most of these physicochemical and kinetic properties were found to be similar to those of the already well-characterized stem bromelain (EC 3.4.22.32). Thus, the immunoaffinity purified crown leaf protease appeared to be closely related to stem bromelain.

  6. Secretory immunity and the bacterial IgA proteases.

    PubMed

    Kornfeld, S J; Plaut, A G

    1981-01-01

    The characteristics and functions of microbial IgA proteases are reviewed. These enzymes represent a structurally heterogeneous group of proteins that are secreted into the extracellular environment by bacteria capable of causing human disease. The IgA proteases, which vary in their requirements for metal ions, are neutral endopeptidases whose role in the infectious process is not known but whose pronounced substrate specificity for human proteins of the IgA1 subclass has repeatedly been demonstrated. As reagents, the IgA proteases are useful in cleaving IgA molecules to yield intact Fc alpha and Fab alpha fragments that will allow the study of the structure and function of the two large regions of IgA immunoglobulin proteins. The role, if any, of these enzymes in promoting infection by pathogenic members of the genera Neisseria, Hemophilus, and Streptococcus is not known, although the secretory immune system is primarily mediated by antibodies of the IgA isotype, among which are IgA1 subclass proteins, and these proteins are susceptible to cleavage by IgA protease. The determination of the role of these enzymes in the pathogenesis of human infection must await clearer understanding of antigenicity and antibody function at secretory sites and of the relative roles of the two subclasses of human IgA in immune defense.

  7. Processing and targeting of the thiol protease aleurain: Progress report

    SciTech Connect

    Rogers, J.C.

    1988-01-01

    This study addresses the processing and targeting of the thiol protease aleurain in monocots. A probe derived from the aleurain cDNA specific for the 5'-most 400 bp (a region encoding the first 140 amino acids of the preprotein hybridized to at least 3 separate elements in the barley genome; only one represented the aleurain gene. In contrast, a probe specific for the remaining 2/23 of the cDNA (representing the protease domain) hybridized to only a single copy sequence. To know if this pattern pertained in other, closely related, monocots, we probed Southern blots of genomic DNA from maize, rye, oats, sorghum, and pearl millet with each probe. In each instance except for maize DNA, the 5' domain probe hybridizes to several fragments in addition to those identified by the protease domain probe. Presumable the darkest hybridization in each represents the fragment carrying the sequences homologous to barley aleurain. The fragments from a given restriction enzyme identified by the protease domain probe in sorghum, millet, and maize, were indistinguishable in size indicating that the gene sequences, as well as flanking DNA, are so well conserved among the group that the location of the hexanucleotide sequences have not diverged. (3 refs., 3 figs.)

  8. Post-translational control of genetic circuits using Potyvirus proteases.

    PubMed

    Fernandez-Rodriguez, Jesus; Voigt, Christopher A

    2016-07-27

    Genetic engineering projects often require control over when a protein is degraded. To this end, we use a fusion between a degron and an inactivating peptide that can be added to the N-terminus of a protein. When the corresponding protease is expressed, it cleaves the peptide and the protein is degraded. Three protease:cleavage site pairs from Potyvirus are shown to be orthogonal and active in exposing degrons, releasing inhibitory domains and cleaving polyproteins. This toolbox is applied to the design of genetic circuits as a means to control regulator activity and degradation. First, we demonstrate that a gate can be constructed by constitutively expressing an inactivated repressor and having an input promoter drive the expression of the protease. It is also shown that the proteolytic release of an inhibitory domain can improve the dynamic range of a transcriptional gate (200-fold repression). Next, we design polyproteins containing multiple repressors and show that their cleavage can be used to control multiple outputs. Finally, we demonstrate that the dynamic range of an output can be improved (8-fold to 190-fold) with the addition of a protease-cleaved degron. Thus, controllable proteolysis offers a powerful tool for modulating and expanding the function of synthetic gene circuits. PMID:27298256

  9. Generic protease detection technology for monitoring periodontal disease.

    PubMed

    Zheng, Xinwei; Cook, Joseph P; Watkinson, Michael; Yang, Shoufeng; Douglas, Ian; Rawlinson, Andrew; Krause, Steffi

    2011-01-01

    Periodontal diseases are inflammatory conditions that affect the supporting tissues of teeth and can lead to destruction of the bone support and ultimately tooth loss if untreated. Progression of periodontitis is usually site specific but not uniform, and currently there are no accurate clinical methods for distinguishing sites where there is active disease progression from sites that are quiescent. Consequently, unnecessary and costly treatment of periodontal sites that are not progressing may occur. Three proteases have been identified as suitable markers for distinguishing sites with active disease progression and quiescent sites: human neutrophil elastase, cathepsin G and MMP8. Generic sensor materials for the detection of these three proteases have been developed based on thin dextran hydrogel films cross-linked with peptides. Degradation of the hydrogel films was monitored using impedance measurements. The target proteases were detected in the clinically relevant range within a time frame of 3 min. Good specificity for different proteases was achieved by choosing appropriate peptide cross-linkers.

  10. THE ROLE OF CYSTEINE PROTEASE IN ALZHEIMER DISEASE

    PubMed Central

    Hasanbasic, Samra; Jahic, Alma; Karahmet, Emina; Sejranic, Asja; Prnjavorac, Besim

    2016-01-01

    Introduction: Cysteine protease are biological catalysts which play a pivotal role in numerous biological reactions in organism. Much of the literature is inscribed to their biochemical significance, distribution and mechanism of action. Many diseases, e.g. Alzheimer’s disease, develop due to enzyme balance disruption. Understanding of cysteine protease’s disbalance is therefor a key to unravel the new possibilities of treatment. Cysteine protease are one of the most important enzymes for protein disruption during programmed cell death. Whether protein disruption is part of cell deaths is not enough clear in any cases. Thereafter, any tissue disruption, including proteolysis, generate more or less inflammation appearance. Review: This review briefly summarizes the current knowledge about pathological mechanism’s that results in AD, with significant reference to the role of cysteine protease in it. Based on the summary, new pharmacological approach and development of novel potent drugs with selective toxicity targeting cysteine protease will be a major challenge in years to come. PMID:27482169

  11. Design, synthesis, and activity of nanocellulosic protease sensors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Here we contrast the molecular assembly, and biochemical utility of nanocellulosic materials prepared from cotton and wood as protease sensors. The cotton-based nanocellulosic substrates were prepared in a variety of ways to produce nanocrystals, films and aerogels, which were derivatized with eithe...

  12. graal: a Drosophila gene coding for several mosaic serine proteases.

    PubMed

    Munier, Anne Isabelle; Medzhitov, Ruslan; Janeway, Charles A; Doucet, Daniel; Capovilla, Maria; Lagueux, Marie

    2004-10-01

    Serine proteases play vital roles in several biological processes such as development and immunity. We have characterized Graal, a large multi-domain serine protease from Drosophila. Graal is spliced in at least three transcripts that are present throughout development. The domains found in Graal proteins are: chitin-binding domains (CBD), scavenger receptor cysteine-rich (SRCR) domains, low density lipoprotein receptor cysteine-rich (LDLR-CR) domains, histidine and proline-rich domains, a NGGYQPP-repeat domain and a serine protease domain. The last 2370 nucleotides of these RNAs are identical and encode a His-rich domain, two SRCR domains, two LDLR-CR domains and a protease domain. The transcription of graal is upregulated after fungal or bacterial infection. Analysis of the Iso1 (y;cn,sp,bw) strain shows that graal transcription is impaired in this fly line due to the insertion of a retrotransposon in the sixth exon. However, no phenotype could be observed consecutive to the absence of graal full length transcripts, particularly in the context of an immune challenge.

  13. Differential protease activity augments polyphagy in Helicoverpa armigera.

    PubMed

    Chikate, Y R; Tamhane, V A; Joshi, R S; Gupta, V S; Giri, A P

    2013-06-01

    Helicoverpa armigera (Lepidoptera: Noctuidae) and other polyphagous agricultural pests are extending their plant host range and emerging as serious agents in restraining crop productivity. Dynamic regulation, coupled with a diversity of digestive and detoxifying enzymes, play a crucial role in the adaptation of polyphagous insects. To investigate the functional intricacy of serine proteases in the development and polyphagy of H. armigera, we profiled the expression of eight trypsin-like and four chymotrypsin-like phylogenetically diverse mRNAs from different life stages of H. armigera reared on nutritionally distinct host plants. These analyses revealed diet- and stage-specific protease expression patterns. The trypsins expressed showed structural variations, which might result in differential substrate specificity and interaction with inhibitors. Protease profiles in the presence of inhibitors and their mass spectrometric analyses revealed insight into their differential activity. These findings emphasize the differential expression of serine proteases and their consequences for digestive physiology in promoting polyphagy in H. armigera. PMID:23432026

  14. Recent trends in protease-catalyzed peptide synthesis.

    PubMed

    Lombard, C; Saulnier, J; Wallach, J M

    2005-10-01

    Enzymatic peptide syntheses may be either thermodynamically- or kinetically-controlled. The former may be catalyzed by any proteases; the latter is limited to serine and cysteine proteases. This methodology is quite stereospecific and avoids side chain protection but is suffering of some drawbacks. Thus, only two industrial processes are by now developed: the production of aspartame and the conversion of porcine into human insulin. However, recent improvements have been carried out in different directions: 1-Search for proteases with high and/or new P'1 and P1 specificities. 2-Protease engineering to promote synthesis towards hydrolysis and to enlarge specificity. 3-Development of mimetic or "inverse" substrates to limit further hydrolysis of synthesized peptide. 4-Change of the physical state of reactants. Three axes have mainly be explored: solid-solid conversion, use of cross-linked enzyme crystals (CLEC) and enzyme immobilization. 5-Modification of experimental conditions. The principal and recent developments deal with: heterogeneous catalysis, synthesis in low water-containing organic solvents, in ionic liquids or at subzero temperatures. This review will illustrate these new orientations with examples described in the recent literature.

  15. Botulinum neurotoxin: a deadly protease with applications to human medicine

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Botulinum neurotoxins (BoNTs) are some of the most potent biological toxins to humans. They are synthesized by the gram-positive, spore-forming bacterium Clostridium botulinum. BoNT is secreted from the bacterium as a ~150 kDa polypeptide which is cleaved by bacterial or host proteases into a ~50 kD...

  16. Protease-activated-receptor-2 affects protease-activated-receptor-1-driven breast cancer.

    PubMed

    Jaber, Mohammad; Maoz, Miriam; Kancharla, Arun; Agranovich, Daniel; Peretz, Tamar; Grisaru-Granovsky, Sorina; Uziely, Beatrice; Bar-Shavit, Rachel

    2014-07-01

    Mammalian protease-activated-receptor-1 and -2 (PAR1 and PAR2) are activated by proteases found in the flexible microenvironment of a tumor and play a central role in breast cancer. We propose in the present study that PAR1 and PAR2 act together as a functional unit during malignant and physiological invasion processes. This notion is supported by assessing pro-tumor functions in the presence of short hairpin; shRNA knocked-down hPar2 or by the use of a truncated PAR2 devoid of the entire cytoplasmic tail. Silencing of hPar2 by shRNA-attenuated thrombin induced PAR1 signaling as recapitulated by inhibiting the assembly of Etk/Bmx or Akt onto PAR1-C-tail, by thrombin-instigated colony formation and invasion. Strikingly, shRNA-hPar2 also inhibited the TFLLRN selective PAR1 pro-tumor functions. In addition, while evaluating the physiological invasion process of placenta extravillous trophoblast (EVT) organ culture, we observed inhibition of both thrombin or the selective PAR1 ligand; TFLLRNPNDK induced EVT invasion by shRNA-hPar2 but not by scrambled shRNA-hPar2. In parallel, when a truncated PAR2 was utilized in a xenograft mouse model, it inhibited PAR1-PAR2-driven tumor growth in vivo. Similarly, it also attenuated the interaction of Etk/Bmx with the PAR1-C-tail in vitro and decreased markedly selective PAR1-induced Matrigel invasion. Confocal images demonstrated co-localization of PAR1 and PAR2 in HEK293T cells over-expressing YFP-hPar2 and HA-hPar1. Co-immuno-precipitation analyses revealed PAR1-PAR2 complex formation but no PAR1-CXCR4 complex was formed. Taken together, our observations show that PAR1 and PAR2 act as a functional unit in tumor development and placenta-uterus interactions. This conclusion may have significant consequences on future breast cancer therapeutic modalities and improved late pregnancy outcome. PMID:24177339

  17. The second decade of 3C technologies: detailed insights into nuclear organization

    PubMed Central

    Denker, Annette; de Laat, Wouter

    2016-01-01

    The relevance of three-dimensional (3D) genome organization for transcriptional regulation and thereby for cellular fate at large is now widely accepted. Our understanding of the fascinating architecture underlying this function is based on microscopy studies as well as the chromosome conformation capture (3C) methods, which entered the stage at the beginning of the millennium. The first decade of 3C methods rendered unprecedented insights into genome topology. Here, we provide an update of developments and discoveries made over the more recent years. As we discuss, established and newly developed experimental and computational methods enabled identification of novel, functionally important chromosome structures. Regulatory and architectural chromatin loops throughout the genome are being cataloged and compared between cell types, revealing tissue invariant and developmentally dynamic loops. Architectural proteins shaping the genome were disclosed, and their mode of action is being uncovered. We explain how more detailed insights into the 3D genome increase our understanding of transcriptional regulation in development and misregulation in disease. Finally, to help researchers in choosing the approach best tailored for their specific research question, we explain the differences and commonalities between the various 3C-derived methods. PMID:27340173

  18. Contemporaneous IUE, EUVE, and High-Energy Observations of 3C 273

    NASA Technical Reports Server (NTRS)

    Ramos, E.; Kafatos, M.; Fruscione, A.; Bruhweiler, F. C.; McHardy, I. M.; Hartman, R. C.; Titarchuk, L. G.; vonMontigny, C.

    1997-01-01

    We present the results of our 1994 January and 1995 January observations of the quasar 3C 273 obtained with the International Ultraviolet Explorer (IUE) and the Extreme-Ultraviolet Explorer (EUVE). These observations were part of a large multiwavelength campaign to observe 3C 273 from radio through gamma-rays. Our 1995 January photometric observations with the EUVE Lexan/B Deep Survey (DS) instrument indicate strong evidence for variability, at a 99% confidence level, during the 12 day observing period. We have utilized ROSAT PSPC soft X-ray power-law models to correlate with EUVE count rates. Besides variations in the normalization level between both observations, our EUV count rates are consistent with a simple power-law model with spectral index alpha approx. 1.77 (F(sub upsilon) proportional to upsilon(sup -alpha) that can be extrapolated from the soft X-rays to the EUV range. The active galactic nucleus 3C 273 is an important blazar to study because in our picture it reveals the presence of both disk and relativistic beam spectral contributions.

  19. Possible light-induced superconductivity in K3C60 at high temperature.

    PubMed

    Mitrano, M; Cantaluppi, A; Nicoletti, D; Kaiser, S; Perucchi, A; Lupi, S; Di Pietro, P; Pontiroli, D; Riccò, M; Clark, S R; Jaksch, D; Cavalleri, A

    2016-02-25

    The non-equilibrium control of emergent phenomena in solids is an important research frontier, encompassing effects such as the optical enhancement of superconductivity. Nonlinear excitation of certain phonons in bilayer copper oxides was recently shown to induce superconducting-like optical properties at temperatures far greater than the superconducting transition temperature, Tc (refs 4-6). This effect was accompanied by the disruption of competing charge-density-wave correlations, which explained some but not all of the experimental results. Here we report a similar phenomenon in a very different compound, K3C60. By exciting metallic K3C60 with mid-infrared optical pulses, we induce a large increase in carrier mobility, accompanied by the opening of a gap in the optical conductivity. These same signatures are observed at equilibrium when cooling metallic K3C60 below Tc (20 kelvin). Although optical techniques alone cannot unequivocally identify non-equilibrium high-temperature superconductivity, we propose this as a possible explanation of our results.

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

    NASA Astrophysics Data System (ADS)

    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.

  1. Ni3C-assisted growth of carbon nanofibres 300 °C by thermal CVD

    NASA Astrophysics Data System (ADS)

    Yu, Bowen; Wang, Shiliang; Zhang, Qiankun; He, Yuehui; Huang, Han; Zou, Jin

    2014-08-01

    Ni-assisted thermal chemical vapor deposition (TCVD) is one of the most common techniques for the growth of carbon nanofibres/nanotubes (CNFs/CNTs). However, some fundamental issues related to the catalytic growth of CNFs/CNTs, such as the low-limit growth temperature, the limiting steps and the state of Ni, are still controversial. Here, we report the growth of CNFs at 300 °C that is the lowest temperature for the growth of CNFs by TCVD using Ni as the catalyst so far. The results showed that the Ni existed in rhombohedral Ni3C, not in the normal form of face-centered cubic Ni, and the C atoms for building the CNFs were precipitated from the (001) planes of the faceted Ni3C nanoparticles. The CNFs are believed to be formed by the decomposition-formation cycle of metastable Ni3C that has a low-limit decomposition temperature of about 300 °C. Our results strongly suggest that TCVD is a valuable tool for the synthesis of CNFs/CNTs at temperatures below 400 °C, which is generally considered as the upper-limit temperature for fabricating complementary metal oxide semiconductor devices but is the low-limit temperature for growing CNFs/CNTs by TCVD at present.

  2. Superconductivity of Rb 3 C 60 : breakdown of the Migdal-Eliashberg theory

    NASA Astrophysics Data System (ADS)

    Cappelluti, E.; Grimaldi, C.; Pietronero, L.; Strässler, S.; Ummarino, G. A.

    2001-06-01

    In this paper, through an exhaustive analysis within the Migdal-Eliashberg theory, we show the incompatibility of experimental data of Rb3C60 with the basic assumptions of the standard theory of superconductivity. For different models of the electron-phonon spectral function α2F(Ω) we solve numerically the Eliashberg equations to find which values of the electron-phonon coupling λ, of the logarithmic phonon frequency and of the Coulomb pseudopotential μ* reproduce the experimental data of Rb3C60. We find that the solutions are essentially independent of the particular shape of α2F(Ω) and that, to explain the experimental data of Rb3C60, one has to resort to extremely large couplings: λ = 3.0+/-0.8. This results differs from the usual partial analyses reported up to now and we claim that this value exceeds the maximum allowed λ compatible with the crystal lattice stability. Moreover, we show quantitatively that the obtained values of λ and strongly violate Migdal's theorem and consequently are incompatible with the Migdal-Eliashberg theory. One has therefore to consider the generalization of the theory of superconductivity in the nonadiabatic regime to account for the experimental properties of fullerides.

  3. Preclinical evaluation of a monoclonal antibody (3C6) specific for prostate-specific membrane antigen

    PubMed Central

    Regino, C.A.S.; Wong, K.J.; Milenic, D.E.; Holmes, E.H.; Garmestani, K.; Choyke, P.L.; Brechbiel, M.W.

    2008-01-01

    Better tumor markers are needed for early diagnosis and staging of prostate cancer, and for monitoring therapeutic response than the currently used prostate specific antigen (PSA). Prostate specific membrane antigen (PSMA) is highly expressed on the surface of prostatic epithelial cells making it a good target for prostate cancer. In this study, mAb 3C6, specific for the extracellular epitope of PSMA, was evaluated both in vitro and in vivo for PSMA-targeting. Immunoreactivity and specificity of mAb 3C6 was evaluated by flow cytometry using prostate cell lines expressing PSMA such as LNCaP and 22Rv1 and a cell line, DU145, that expresses very little PSMA. 3C6 was conjugated with the acyclic CHX-A” DTPA chelate, radiolabeled with 111In, and its in vitro and in vivo properties were assessed. The biodistribution of the radioimmunoconjugate evaluated in athymic mice bearing xenografts of three human prostate carcinoma cell lines shows high uptake after 72 hr in LNCaP tumors (%ID/g 22.93 ± 6.32) and 22Rv1 (%ID/g 10.44 ± 2.32) in contrast to low uptake by the DU145 tumors (%ID/g 4.27 ± 0.37). Planar γ-scintigraphic images obtained for xenografted tumor bearing mice demonstrated targeting for PSMA positive tumors suggesting possible applications in imaging and for targeted radiation therapy. PMID:20047017

  4. Spectroscopy and optically stimulated luminescence of Al2O3:C using time-resolved measurements

    NASA Astrophysics Data System (ADS)

    Yukihara, E. G.; McKeever, S. W. S.

    2006-10-01

    This paper reports the observation of ultraviolet (UV) emission at 335nm in the optically stimulated luminescence (OSL) of carbon-doped aluminum oxide (Al2O3:C) and presents results on the investigation of the OSL properties of this band, including its dose response, time dependence after irradiation, and dependence of the OSL signal on the type of radiation. Time-resolved OSL measurements were used to separate the UV emission band from the dominant OSL emission band of Al2O3:C, namely, the F-center luminescence at 420nm. A comparison of the OSL properties of the UV and F-center emission bands is important for various dosimetric applications because the relative contribution of the UV and F-center emissions to the OSL signal varies with readout technique and optical filters used in the readout equipment. The UV emission band is found to show an ionization density dependence that is different from the dependence observed for the F-center emission, and an increase in intensity with time elapsed after beta irradiation. These results are relevant for OSL dosimetry of radiation fields containing heavy charged particles, such as the space radiation field and the secondary fields created by interactions of matter with energetic neutrons, as well as for understanding of the basic OSL mechanism in Al2O3:C.

  5. A short-time fading study of Al2O3:C

    NASA Astrophysics Data System (ADS)

    Nascimento, L. F.; Vanhavere, F.; Silva, E. H.; Deene, Y. De

    2015-01-01

    This paper studies the short-time fading from Al2O3:C by measuring optically stimulated luminescence (OSL) signals (Total OSL: TOSL, and Peak OSL: POSL) from droplets and Luxel™ pellets. The influence of various bleaching regimes (blue, green and white) and light power is compared. The fading effect is the decay of the OSL signal in the dark at room temperature. Al2O3:C detectors were submitted to various bleaching regimes, irradiated with a reference dose and read out after different time spans. Investigations were carried out using 2 mm size droplet detectors, made of thin Al2O3:C powder mixed with a photocured polymer. Tests were compared to Luxel™-type detectors (Landauer Inc.). Short-time post-irradiation fading is present in OSL results (TOSL and POSL) droplets for time spans up to 200 s. The effect of short-time fading can be lowered/removed when treating the detectors with high-power and/or long time bleaching regimes; this result was observed in both TOSL and POSL from droplets and Luxel™.

  6. An HST Proper-motion Study of the Large-scale Jet of 3C273

    NASA Astrophysics Data System (ADS)

    Meyer, Eileen T.; Sparks, William B.; Georganopoulos, Markos; Anderson, Jay; van der Marel, Roeland; Biretta, John; Sohn, Sangmo Tony; Chiaberge, Marco; Perlman, Eric; Norman, Colin

    2016-02-01

    The radio galaxy 3C 273 hosts one of the nearest and best-studied powerful quasar jets. Having been imaged repeatedly by the Hubble Space Telescope (HST) over the past twenty years, it was chosen for an HST program to measure proper motions in the kiloparsec-scale resolved jets of nearby radio-loud active galaxies. The jet in 3C 273 is highly relativistic on sub-parsec scales, with apparent proper motions up to 15c observed by very long baseline interferometry. In contrast, we find that the kiloparsec-scale knots are compatible with being stationary, with a mean speed of -0.2 ± 0.5c over the whole jet. Assuming the knots are packets of moving plasma, an upper limit of 1c implies a bulk Lorentz factor Γ < 2.9. This suggests that the jet has either decelerated significantly by the time it reaches the kiloparsec scale, or that the knots in the jet are standing shock features. The second scenario is incompatible with the inverse Compton off the Cosmic Microwave Background (IC/CMB) model for the X-ray emission of these knots, which requires the knots to be in motion, but IC/CMB is also disfavored in the first scenario due to energetic considerations, in agreement with the recent finding of Meyer & Georganopoulos which ruled out the IC/CMB model for the X-ray emission of 3C 273 via gamma-ray upper limits.

  7. The far-infrared emission of the radio-loud quasar 3C 318

    NASA Astrophysics Data System (ADS)

    Podigachoski, P.; Barthel, P. D.; Peletier, R. F.; Steendam, S.

    2016-01-01

    3C 318, a radio-loud quasar at z = 1.574, is a subgalactic-sized radio source, and a good test-bed for the interplay between black hole and galaxy growth in the high-z Universe. Based on its IRAS, ISO, and SCUBA detections, it has long been considered as one of the most intrinsically luminous (LIR > 1013 L⊙) infrared sources in the Universe. Recent far-infrared data from the Herschel Space Observatory reveal that most of the flux associated with 3C 318, measured with earlier instruments, in fact comes from a bright nearby source. Optical imaging and spectroscopy show that this infrared-bright source is a strongly star-forming pair of interacting galaxies at z = 0.35. Adding existing Spitzer and SDSS photometry, we perform a spectral energy distribution analysis of the pair, and find that it has a combined infrared luminosity of LIR = 1.5 × 1012 L⊙, comparable to other intermediate-redshift ultra-luminous infrared galaxies studied with Herschel. Isolating the emission from 3C 318's host, we robustly constrain the level of star formation to a value a factor of three lower than that published earlier, which is more in line with the star formation activity found in other Herschel-detected 3CR objects at similar redshift.

  8. The second decade of 3C technologies: detailed insights into nuclear organization.

    PubMed

    Denker, Annette; de Laat, Wouter

    2016-06-15

    The relevance of three-dimensional (3D) genome organization for transcriptional regulation and thereby for cellular fate at large is now widely accepted. Our understanding of the fascinating architecture underlying this function is based on microscopy studies as well as the chromosome conformation capture (3C) methods, which entered the stage at the beginning of the millennium. The first decade of 3C methods rendered unprecedented insights into genome topology. Here, we provide an update of developments and discoveries made over the more recent years. As we discuss, established and newly developed experimental and computational methods enabled identification of novel, functionally important chromosome structures. Regulatory and architectural chromatin loops throughout the genome are being cataloged and compared between cell types, revealing tissue invariant and developmentally dynamic loops. Architectural proteins shaping the genome were disclosed, and their mode of action is being uncovered. We explain how more detailed insights into the 3D genome increase our understanding of transcriptional regulation in development and misregulation in disease. Finally, to help researchers in choosing the approach best tailored for their specific research question, we explain the differences and commonalities between the various 3C-derived methods. PMID:27340173

  9. Structural Domain Switching by Magnetic Fields in RAl3C3 (R = Rare Earth)

    NASA Astrophysics Data System (ADS)

    Matsumura, Takeshi; Hayashi, Yuya; Takai, Shun; Otsubo, Toru; Matsuda, Saori; Ochiai, Akira

    2015-11-01

    Hexagonal-to-orthorhombic structural transitions in RAl3C3 have been studied by X-ray diffraction for R = Yb, Tm, Er, Dy, and Lu. We report how orthorhombic domains are controlled by adjusting external magnetic fields. It is shown that orthorhombic domains in RAl3C3 can be aligned by the field when the R ion is magnetic. It is also shown that a single-domain state can be switched to another single-domain state by the field even in paramagnetic states with small induced moments. For R = Lu, however, it was not possible to change the multidomain state by a field of up to 70 kOe. We discuss that domain selection and switching are caused by magnetic anisotropies in the orthorhombic phase of RAl3C3. We also propose an idea to explain the field-induced antiferromagnetism revealed by Khalyavin et al. [Phys. Rev. B 87, 220406(R) (2013)].

  10. Possible light-induced superconductivity in K3C60 at high temperature.

    PubMed

    Mitrano, M; Cantaluppi, A; Nicoletti, D; Kaiser, S; Perucchi, A; Lupi, S; Di Pietro, P; Pontiroli, D; Riccò, M; Clark, S R; Jaksch, D; Cavalleri, A

    2016-02-25

    The non-equilibrium control of emergent phenomena in solids is an important research frontier, encompassing effects such as the optical enhancement of superconductivity. Nonlinear excitation of certain phonons in bilayer copper oxides was recently shown to induce superconducting-like optical properties at temperatures far greater than the superconducting transition temperature, Tc (refs 4-6). This effect was accompanied by the disruption of competing charge-density-wave correlations, which explained some but not all of the experimental results. Here we report a similar phenomenon in a very different compound, K3C60. By exciting metallic K3C60 with mid-infrared optical pulses, we induce a large increase in carrier mobility, accompanied by the opening of a gap in the optical conductivity. These same signatures are observed at equilibrium when cooling metallic K3C60 below Tc (20 kelvin). Although optical techniques alone cannot unequivocally identify non-equilibrium high-temperature superconductivity, we propose this as a possible explanation of our results. PMID:26855424

  11. Recombining plasma in the gamma-ray-emitting mixed-morphology supernova remnant 3C 391

    SciTech Connect

    Ergin, T.; Sezer, A.; Saha, L.; Majumdar, P.; Chatterjee, A.; Bayirli, A.; Ercan, E. N.

    2014-07-20

    A group of middle-aged mixed-morphology (MM) supernova remnants (SNRs) interacting with molecular clouds (MCs) has been discovered to be strong GeV gamma-ray emitters by the Large Area Telescope (LAT) on board the Fermi Gamma-Ray Space Telescope (Fermi-LAT). The recent observations of the Suzaku X-ray satellite have revealed that some of these interacting gamma-ray-emitting SNRs, such as IC443, W49B, W44, and G359.1-0.5, have overionized plasmas. 3C 391 (G31.9+0.0) is another Galactic MM SNR interacting with MCs. It was observed in GeV gamma rays by Fermi-LAT as well as in the 0.3-10.0 keV X-ray band by Suzaku. In this work, 3C 391 was detected in GeV gamma rays with a significance of ∼18σ and we showed that the GeV emission is point-like in nature. The GeV gamma-ray spectrum was shown to be best explained by the decay of neutral pions assuming that the protons follow a broken power-law distribution. We revealed radiative recombination structures of silicon and sulfur from 3C 391 using Suzaku data. In this paper, we discuss the possible origin of this type of radiative plasma and hadronic gamma rays.

  12. Ni3Mo3C as anode catalyst for high-performance microbial fuel cells.

    PubMed

    Zeng, Li-Zhen; Zhao, Shao-Fei; Li, Wei-Shan

    2015-03-01

    Ni3Mo3C was prepared by a modified organic colloid method and explored as anode catalyst for high-performance microbial fuel cell (MFC) based on Klebsiella pneumoniae (K. pneumoniae). The prepared sample was characterized by X-ray diffraction (XRD), scanning electron microscope (SEM), and Brunauer-Emmett-Teller (BET). The activity of the sample as anode catalyst for MFC based on K. pneumoniae was investigated by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and polarization curve measurement. The results show that the adding of nickel in Mo2C increases the BET surface area of Mo2C and improves the electrocatalytic activity of Mo2C towards the oxidation of microbial fermentation products. The power density of MFC with 3 mg cm(-2) Ni3Mo3C anode is far higher than that of the MFC with carbon felt as anode without any catalyst, which is 19 % higher than that of Mo2C anode and produced 62 % as much as that of Pt anode, indicating that Ni3Mo3C is comparative to noble metal platinum as anode electrocatalyst for MFCs by increasing the loading.

  13. Radio jets and bridges in the classical double sources 3C388 and 0816 + 526

    NASA Astrophysics Data System (ADS)

    Burns, J. O.; Christiansen, W. A.

    1980-09-01

    Full-synthesis high-resolution (1 arcsec) observations of the classical double radio sources 0816 + 526 and 3C 388 are reported. The radio galaxies were observed at 4885 MHz using the Very Large Array with baselines ranging from 60 m to 18 km. Contour maps of the total intensity distribution reveal the presence of sharp leading edges indicative of deceleration in 0816 + 526, together with steep transverse boundaries of the two components consistent with constant emissivity-filled cylinders. 3C 388 is found to contain a bright radio jet embedded within the southwestern lobe, with a gap in the radio emission between the galactic nucleus and the jet. The structure of 0816 + 526 is attributed to the penetration of the radio source into an enhanced background density, while 3C 388 is interpreted as an interaction between an intermittent beam and/or fresh plasmoid and the boundaries of an evacuated cavity or bubble. In addition, observations clearly illustrate the variety of structures that may exist in radio galaxies classified simply as classical doubles.

  14. Multi-Epoch Multiwavelength Spectra and Models for Blazar 3C 279

    NASA Technical Reports Server (NTRS)

    Hartman, R. C.; Boettcher, M.; Aldering, G.; Aller, H.; Aller, M.; Backman, D. E.; Balonek, T. J.; Bertsch, D. L.; Bloom, S. D.; Bock, H.; White, Nicholas E. (Technical Monitor)

    2001-01-01

    Of the blazars detected by EGRET in GeV gamma-rays, 3C 279 is not only the best-observed by EGRET, but also one of the best-monitored at lower frequencies. We have assembled eleven spectra, from GHz radio through GeV gamma-rays, from the time intervals of EGRET observations. Although some of the data have appeared in previous publications, most are new, including data taken during the high states in early 1999 and early 2000. All of the spectra show substantial gamma-ray contribution to the total luminosity of the object; in a high state, the gamma-ray luminosity dominates over that at all other frequencies by a factor of more than 10. There is no clear pattern of time correlation; different bands do not always rise and fall together, even in the optical, X-ray, and gamma-ray bands. The spectra are modeled using a leptonic jet, with combined synchrotron self-Compton + external Compton gamma-ray production. Spectral variability of 3C 279 is consistent with variations of the bulk Lorentz factor of the jet, accompanied by changes in the spectral shape of the electron distribution. Our modeling results are consistent with the UV spectrum of 3C 279 being dominated by accretion disk radiation during times of low gamma-ray intensity.

  15. The second decade of 3C technologies: detailed insights into nuclear organization.

    PubMed

    Denker, Annette; de Laat, Wouter

    2016-06-15

    The relevance of three-dimensional (3D) genome organization for transcriptional regulation and thereby for cellular fate at large is now widely accepted. Our understanding of the fascinating architecture underlying this function is based on microscopy studies as well as the chromosome conformation capture (3C) methods, which entered the stage at the beginning of the millennium. The first decade of 3C methods rendered unprecedented insights into genome topology. Here, we provide an update of developments and discoveries made over the more recent years. As we discuss, established and newly developed experimental and computational methods enabled identification of novel, functionally important chromosome structures. Regulatory and architectural chromatin loops throughout the genome are being cataloged and compared between cell types, revealing tissue invariant and developmentally dynamic loops. Architectural proteins shaping the genome were disclosed, and their mode of action is being uncovered. We explain how more detailed insights into the 3D genome increase our understanding of transcriptional regulation in development and misregulation in disease. Finally, to help researchers in choosing the approach best tailored for their specific research question, we explain the differences and commonalities between the various 3C-derived methods.

  16. Ni3C-assisted growth of carbon nanofibres 300 °C by thermal CVD.

    PubMed

    Yu, Bowen; Wang, Shiliang; Zhang, Qiankun; He, Yuehui; Huang, Han; Zou, Jin

    2014-08-15

    Ni-assisted thermal chemical vapor deposition (TCVD) is one of the most common techniques for the growth of carbon nanofibres/nanotubes (CNFs/CNTs). However, some fundamental issues related to the catalytic growth of CNFs/CNTs, such as the low-limit growth temperature, the limiting steps and the state of Ni, are still controversial. Here, we report the growth of CNFs at 300 °C; that is the lowest temperature for the growth of CNFs by TCVD using Ni as the catalyst so far. The results showed that the Ni existed in rhombohedral Ni3C, not in the normal form of face-centered cubic Ni, and the C atoms for building the CNFs were precipitated from the (001) planes of the faceted Ni3C nanoparticles. The CNFs are believed to be formed by the decomposition-formation cycle of metastable Ni3C that has a low-limit decomposition temperature of about 300 °C. Our results strongly suggest that TCVD is a valuable tool for the synthesis of CNFs/CNTs at temperatures below 400 °C, which is generally considered as the upper-limit temperature for fabricating complementary metal oxide semiconductor devices but is the low-limit temperature for growing CNFs/CNTs by TCVD at present. PMID:25053710

  17. Detailed study of the microwave emission of the supernova remnant 3C 396

    NASA Astrophysics Data System (ADS)

    Cruciani, A.; Battistelli, E. S.; Carretti, E.; de Bernardis, P.; Genova-Santos, R.; Masi, S.; Mason, B.; Perera, D.; Piacentini, F.; Reach, B.; Rubino-Martin, J. A.

    2016-07-01

    We have observed the supernova remnant 3C 396 in the microwave region using the Parkes 64-m telescope. Observations have been made at 8.4, 13.5, and 18.6 GHz and in polarization at 21.5 GHz. We have used data from several other observatories, including previously unpublished observations performed by the Green Bank Telescope at 31.2 GHz, to investigate the nature of the microwave emission of 3C 396. Results show a spectral energy distribution dominated by a single component power-law emission with α = (-0.364 ± 0.017). Data do not favour the presence of anomalous microwave emission coming from the source. Polarized emission at 21.5 GHz is consistent with synchrotron-dominated emission. We present microwave maps and correlate them with infrared (IR) maps in order to characterize the interplay between thermal dust and microwave emission. IR versus microwave TT plots reveal poor correlation between mid-IR and microwave emission from the core of the source. On the other hand, a correlation is detected in the tail emission of the outer shell of 3C 396, which could be ascribed to Galactic contamination.

  18. Carbon Monoxide Observations Toward the Supernova Remnant 3C 391: Interaction Confirmed

    NASA Astrophysics Data System (ADS)

    Wilner, D. J.; Reynolds, S. P.; Moffett, D. A.

    1996-05-01

    We present observations of the J = 1 -> 0 transition of CO toward the radio-bright supernova remnant 3C 391 made with the NRAO 12-m telescope.(*) Our earlier radio continuum observations of 3C 391 showed a morphology strongly suggestive of evolution near a density discontinuity in the external medium. In this interpretation, the supernova went off near the edge of a moderately dense molecular cloud, and after a few hundred to a thousand years the blast wave broke out of the edge of the cloud to emerge as a larger, lower surface-brightness extension to the SNR. Survey data indicated nearby CO at appropriate velocities but direct evidence for interaction was lacking. Our new observations strongly support the interaction picture. CO contours drop steeply at the location of the inner edge of the SNR radio emission; the ``blowout'' extension is in the direction of steepest decrease of CO contours. Extensions on the bright limb of radio continuum perfectly match the edges of a strong CO condensation. A consistent picture then involves the SNR blast wave eating its way into the molecular cloud, destroying CO as it goes. The data also indicate directly that shock acceleration of electrons at least to 10 GeV or so is possible even for a blast wave encountering dense neutral material. We describe the quantitative implications of the interaction picture for 3C 391 and for SNRs in general. (*) The NRAO is operated by Associated Universities, Inc., under a cooperative agreement with the National Science Foundation.

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

    PubMed

    Kim, Jeong Hee; Lee, Min Jun; Hwang, Inhwan; Hwang, Hyun Jin

    2013-01-01

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

  20. KIF3C and KIF3A Form a Novel Neuronal Heteromeric Kinesin That Associates with Membrane Vesicles

    PubMed Central

    Muresan, Virgil; Abramson, Tatiana; Lyass, Asya; Winter, Dirk; Porro, Elena; Hong, Filbert; Chamberlin, Nancy L.; Schnapp, Bruce J.

    1998-01-01

    We have cloned from rat brain the cDNA encoding an 89,828-Da kinesin-related polypeptide KIF3C that is enriched in brain, retina, and lung. Immunocytochemistry of hippocampal neurons in culture shows that KIF3C is localized to cell bodies, dendrites, and, in lesser amounts, to axons. In subcellular fractionation experiments, KIF3C cofractionates with a distinct population of membrane vesicles. Native KIF3C binds to microtubules in a kinesin-like, nucleotide-dependent manner. KIF3C is most similar to mouse KIF3B and KIF3A, two closely related kinesins that are normally present as a heteromer. In sucrose density gradients, KIF3C sediments at two distinct densities, suggesting that it may be part of two different multimolecular complexes. Immunoprecipitation experiments show that KIF3C is in part associated with KIF3A, but not with KIF3B. Unlike KIF3B, a significant portion of KIF3C is not associated with KIF3A. Consistent with these biochemical properties, the distribution of KIF3C in the CNS has both similarities and differences compared with KIF3A and KIF3B. These results suggest that KIF3C is a vesicle-associated motor that functions both independently and in association with KIF3A. PMID:9487132

  1. The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

    PubMed

    Beers, Eric P; Jones, Alan M; Dickerman, Allan W

    2004-01-01

    The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

  2. Structural Mechanisms of Inactivation in Scabies Mite Serine Protease Paralogues

    SciTech Connect

    Fischer, Katja; Langendorf, Christopher G.; Irving, James A.; Reynolds, Simone; Willis, Charlene; Beckham, Simone; Law, Ruby H.P.; Yang, Sundy; Bashtannyk-Puhalovich, Tanya A.; McGowan, Sheena; Whisstock, James C.; Pike, Robert N.; Kemp, David J.; Buckle, Ashley M.

    2009-08-07

    The scabies mite (Sarcoptes scabiei) is a parasite responsible for major morbidity in disadvantaged communities and immuno-compromised patients worldwide. In addition to the physical discomfort caused by the disease, scabies infestations facilitate infection by Streptococcal species via skin lesions, resulting in a high prevalence of rheumatic fever/heart disease in affected communities. The scabies mite produces 33 proteins that are closely related to those in the dust mite group 3 allergen and belong to the S1-like protease family (chymotrypsin-like). However, all but one of these molecules contain mutations in the conserved active-site catalytic triad that are predicted to render them catalytically inactive. These molecules are thus termed scabies mite inactivated protease paralogues (SMIPPs). The precise function of SMIPPs is unclear; however, it has been suggested that these proteins might function by binding and protecting target substrates from cleavage by host immune proteases, thus preventing the host from mounting an effective immune challenge. In order to begin to understand the structural basis for SMIPP function, we solved the crystal structures of SMIPP-S-I1 and SMIPP-S-D1 at 1.85 {angstrom} and 2.0 {angstrom} resolution, respectively. Both structures adopt the characteristic serine protease fold, albeit with large structural variations over much of the molecule. In both structures, mutations in the catalytic triad together with occlusion of the S1 subsite by a conserved Tyr200 residue is predicted to block substrate ingress. Accordingly, we show that both proteases lack catalytic function. Attempts to restore function (via site-directed mutagenesis of catalytic residues as well as Tyr200) were unsuccessful. Taken together, these data suggest that SMIPPs have lost the ability to bind substrates in a classical 'canonical' fashion, and instead have evolved alternative functions in the lifecycle of the scabies mite.

  3. Nematicidal Bacteria Associated to Pinewood Nematode Produce Extracellular Proteases

    PubMed Central

    Francisco, Romeu; Verissimo, Paula; Santos, Susana S.; Fonseca, Luís; Abrantes, Isabel M. O.; Morais, Paula V.

    2013-01-01

    Bacteria associated with the nematode Bursaphelenchus xylophilus, a pathogen of trees and the causal agent of pine wilt disease (PWD) may play a role in the disease. In order to evaluate their role (positive or negative to the tree), strains isolated from the track of nematodes from infected Pinus pinaster trees were screened, in vitro, for their nematicidal potential. The bacterial products, from strains more active in killing nematodes, were screened in order to identify and characterize the nematicidal agent. Forty-seven strains were tested and, of these, 21 strains showed capacity to produce extracellular products with nematicidal activity. All Burkholderia strains were non-toxic. In contrast, all Serratia strains except one exhibited high toxicity. Nematodes incubated with Serratia strains showed, by SEM observation, deposits of bacteria on the nematode cuticle. The most nematicidal strain, Serratia sp. A88copa13, produced proteases in the supernatant. The use of selective inhibitors revealed that a serine protease with 70 kDa was majorly responsible for the toxicity of the supernatant. This extracellular serine protease is different phylogenetically, in size and biochemically from previously described proteases. Nematicidal assays revealed differences in nematicidal activity of the proteases to different species of Bursaphelenchus, suggesting its usefulness in a primary screen of the nematodes. This study offers the basis for further investigation of PWD and brings new insights on the role bacteria play in the defense of pine trees against B. xylophilus. Understanding all the factors involved is important in order to develop strategies to control B. xylophilus dispersion. PMID:24244546

  4. Serine protease activation of near-silent epithelial Na+ channels.

    PubMed

    Caldwell, Ray A; Boucher, Richard C; Stutts, M Jackson

    2004-01-01

    The regulation of epithelial Na+ channel (ENaC) function is critical for normal salt and water balance. This regulation is achieved through cell surface insertion/retrieval of channels, by changes in channel open probability (Po), or through a combination of these processes. Epithelium-derived serine proteases, including channel activating protease (CAP) and prostasin, regulate epithelial Na+ transport, but the molecular mechanism is unknown. We tested the hypothesis that extracellular serine proteases activate a near-silent ENaC population resident in the plasma membrane. Single-channel events were recorded in outside-out patches from fibroblasts (NIH/3T3) stably expressing rat alpha-, beta-, and gamma-subunits (rENaC), before and during exposure to trypsin, a serine protease homologous to CAP and prostasin. Under baseline conditions, near-silent patches were defined as having rENaC activity (NPo) < 0.03, where N is the number of channels. Within 1-5 min of 3 microg/ml bath trypsin superfusion, NPo increased approximately 66-fold (n = 7). In patches observed to contain a single functional channel, trypsin increased Po from 0.02 +/- 0.01 to 0.57 +/- 0.03 (n = 3, mean +/- SE), resulting from the combination of an increased channel open time and decreased channel closed time. Catalytic activity was required for activation of near-silent ENaC. Channel conductance and the Na+/Li+ current ratio with trypsin were similar to control values. Modulation of ENaC Po by endogenous epithelial serine proteases is a potentially important regulator of epithelial Na+ transport, distinct from the regulation achieved by hormone-induced plasma membrane insertion of channels. PMID:12967915

  5. Test results for the Oasis 3C high performance water-pumping windmill

    SciTech Connect

    Eggleston, D.M.

    1997-12-31

    The WINDTech International, L.L.C. Oasis 3C, a 3 m diameter, high-performance water-pumping windmill, was tested at the DME Engineering Wind Test Site just south of Midland, Texas from August through December, 1996. This machine utilizes a 3:1 gearbox with rotating counterweights, similar to a conventional oilfield pumping unit, driven by a multibladed rotor. The rotating counterweight system balances most of the pumping loads and reduces gear loads and starting torque by a factor of at least two and often by a factor of four or more. The torque reduction substantially extends gear and bearing life, and reduces wind speeds required for starting by 30 to 50% or more. The O3C was tested pumping from a quiescent fluid depth of 12.2 m (40 ft) from a 28.3 m (93 ft)-deep well, with additional pumping depth simulated using a pressure regulator valve system. A 9.53 cm (3.75 in.) diameter Harbison-Fischer seal-less single-acting piston pump was used to eliminate pump seal friction as a variable, and standard O3C stroke lengths of 30.5 and 15.2 cm (12 and 6 inches) were used. The regulator spring was set to give a maximum stroke rate of 33 strokes per minute. The water pumped was returned to the well after flowing through a settling tank. The tests were performed in accordance with AWEA WECS testing standards. Instrumentation provided 16 channels of data to accurately measure machine performance, including starting wind speeds, flow rates, O3C azimuth, tail furl angle, wind direction tracking errors, RPM, sucker rod loads, and other variables. The most significant performance data is summarized herein. A mathematical model of machine performance was developed that fairly accurately predicts performance for each of three test conditions. The results verify that the O3C is capable of pumping water at wind speeds from 30% to more than 50% lower than comparable un-counterbalanced units.

  6. Genome-wide survey of prokaryotic serine proteases: Analysis of distribution and domain architectures of five serine protease families in prokaryotes

    PubMed Central

    Tripathi, Lokesh P; Sowdhamini, R

    2008-01-01

    Background Serine proteases are one of the most abundant groups of proteolytic enzymes found in all the kingdoms of life. While studies have established significant roles for many prokaryotic serine proteases in several physiological processes, such as those associated with metabolism, cell signalling, defense response and development, functional associations for a large number of prokaryotic serine proteases are relatively unknown. Current analysis is aimed at understanding the distribution and probable biological functions of the select serine proteases encoded in representative prokaryotic organisms. Results A total of 966 putative serine proteases, belonging to five families, were identified in the 91 prokaryotic genomes using various sensitive sequence search techniques. Phylogenetic analysis reveals several species-specific clusters of serine proteases suggesting their possible involvement in organism-specific functions. Atypical phylogenetic associations suggest an important role for lateral gene transfer events in facilitating the widespread distribution of the serine proteases in the prokaryotes. Domain organisations of the gene products were analysed, employing sensitive sequence search methods, to infer their probable biological functions. Trypsin, subtilisin and Lon protease families account for a significant proportion of the multi-domain representatives, while the D-Ala-D-Ala carboxypeptidase and the Clp protease families are mostly single-domain polypeptides in prokaryotes. Regulatory domains for protein interaction, signalling, pathogenesis, cell adhesion etc. were found tethered to the serine protease domains. Some domain combinations (such as S1-PDZ; LON-AAA-S16 etc.) were found to be widespread in the prokaryotic lineages suggesting a critical role in prokaryotes. Conclusion Domain architectures of many serine proteases and their homologues identified in prokaryotes are very different from those observed in eukaryotes, suggesting distinct roles

  7. Crystal structure of an intracellular protease from Pyrococcus horikoshii at 2-Å resolution

    PubMed Central

    Du, Xinlin; Choi, In-Geol; Kim, Rosalind; Wang, Weiru; Jancarik, Jaru; Yokota, Hisao; Kim, Sung-Hou

    2000-01-01

    The intracellular protease from Pyrococcus horikoshii (PH1704) and PfpI from Pyrococcus furiosus are members of a class of intracellular proteases that have no sequence homology to any other known protease family. We report the crystal structure of PH1704 at 2.0-Å resolution. The protease is tentatively identified as a cysteine protease based on the presence of cysteine (residue 100) in a nucleophile elbow motif. In the crystal, PH1704 forms a hexameric ring structure, and the active sites are formed at the interfaces between three pairs of monomers. PMID:11114201

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

    ERIC Educational Resources Information Center

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

    2011-01-01

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

  9. Cloning, expression and activity analysis of a novel fibrinolytic serine protease from Arenicola cristata

    NASA Astrophysics Data System (ADS)

    Zhao, Chunling; Ju, Jiyu

    2015-06-01

    The full-length cDNA of a protease gene from a marine annelid Arenicola cristata was amplified through rapid amplification of cDNA ends technique and sequenced. The size of the cDNA was 936 bp in length, including an open reading frame encoding a polypeptide of 270 amino acid residues. The deduced amino acid sequnce consisted of pro- and mature sequences. The protease belonged to the serine protease family because it contained the highly conserved sequence GDSGGP. This protease was novel as it showed a low amino acid sequence similarity (< 40%) to other serine proteases. The gene encoding the active form of A. cristata serine protease was cloned and expressed in E. coli. Purified recombinant protease in a supernatant could dissolve an artificial fibrin plate with plasminogen-rich fibrin, whereas the plasminogen-free fibrin showed no clear zone caused by hydrolysis. This result suggested that the recombinant protease showed an indirect fibrinolytic activity of dissolving fibrin, and was probably a plasminogen activator. A rat model with venous thrombosis was established to demonstrate that the recombinant protease could also hydrolyze blood clot in vivo. Therefore, this recombinant protease may be used as a thrombolytic agent for thrombosis treatment. To our knowledge, this study is the first of reporting the fibrinolytic serine protease gene in A. cristata.

  10. Proteases of Wood Rot Fungi with Emphasis on the Genus Pleurotus

    PubMed Central

    Inácio, Fabíola Dorneles; Ferreira, Roselene Oliveira; de Araujo, Caroline Aparecida Vaz; Brugnari, Tatiane; Castoldi, Rafael; Peralta, Rosane Marina; de Souza, Cristina Giatti Marques

    2015-01-01

    Proteases are present in all living organisms and they play an important role in physiological conditions. Cell growth and death, blood clotting, and immune defense are all examples of the importance of proteases in maintaining homeostasis. There is growing interest in proteases due to their use for industrial purposes. The search for proteases with specific characteristics is designed to reduce production costs and to find suitable properties for certain industrial sectors, as well as good producing organisms. Ninety percent of commercialized proteases are obtained from microbial sources and proteases from macromycetes have recently gained prominence in the search for new enzymes with specific characteristics. The production of proteases from saprophytic basidiomycetes has led to the identification of various classes of proteases. The genus Pleurotus has been extensively studied because of its ligninolytic enzymes. The characteristics of this genus are easy cultivation techniques, high yield, low nutrient requirements, and excellent adaptation. There are few studies in the literature about proteases of Pleurotus spp. This review gathers together information about proteases, especially those derived from basidiomycetes, and aims at stimulating further research about fungal proteases because of their physiological importance and their application in various industries such as biotechnology and medicine. PMID:26180792

  11. Yeast Endoplasmic Reticulum Sequestration Screening for the Engineering of Proteases from Libraries Expressed in Yeast.

    PubMed

    Yi, Li; Taft, Joseph M; Li, Qing; Gebhard, Mark C; Georgiou, George; Iverson, Brent L

    2015-01-01

    There is significant interest in engineering proteases with desired proteolytic properties. We describe a high-throughput fluorescence-activated cell sorting (FACS) assay for detecting altered proteolytic activity of protease in yeast, at the single cell level. This assay relies on coupling yeast endoplasmic reticulum (ER) retention, yeast surface display, and FACS analysis. The method described here allows facile screening of large libraries, and of either protease or substrate variants, including the screening of protease libraries against substrate libraries. We demonstrate the application of this technique in the screening of libraries of Tobacco Etch Virus protease (TEV-P) for altered proteolytic activities. In addition, the generality of this method is also validated by other proteases such as human granzyme K and the hepatitis C virus protease, and the human Abelson tyrosine kinase. PMID:26060071

  12. Production of plant proteases in vivo and in vitro--a review.

    PubMed

    González-Rábade, Nuria; Badillo-Corona, Jesús Agustín; Aranda-Barradas, Juan Silvestre; Oliver-Salvador, María Del Carmen

    2011-01-01

    In the latest two decades, the interest received by plant proteases has increased significantly. Plant enzymes such as proteases are widely used in medicine and the food industry. Some proteases, like papain, bromelain and ficin are used in various processes such as brewing, meat softening, milk-clotting, cancer treatment, digestion and viral disorders. These enzymes can be obtained from their natural source or through in vitro cultures, in order to ensure a continuous source of plant enzymes. The focus of this review will be the production of plant proteases both in vivo and in vitro, with particular emphasis on the different types of commercially important plant proteases that have been isolated and characterized from naturally grown plants. In vitro approaches for the production of these proteases is also explored, focusing on the techniques that do not involve genetic transformation of the plants and the attempts that have been made in order to enhance the yield of the desired proteases. PMID:21889977

  13. Redefining the concept of protease-activated receptors: cathepsin S evokes itch via activation of Mrgprs

    PubMed Central

    Reddy, Vemuri B.; Sun, Shuohao; Azimi, Ehsan; Elmariah, Sarina B.; Dong, Xinzhong; Lerner, Ethan A.

    2015-01-01

    Sensory neurons expressing Mas-related G protein coupled receptors (Mrgprs) mediate histamine-independent itch. We show that the cysteine protease cathepsin S activates MrgprC11 and evokes receptor-dependent scratching in mice. In contrast to its activation of conventional protease-activated receptors, cathepsin S mediated activation of MrgprC11 did not involve the generation of a tethered ligand. We demonstrate further that different cysteine proteases selectively activate specific mouse and human Mrgpr family members. This expansion of our understanding by which proteases interact with GPCRs redefines the concept of what constitutes a protease-activated receptor. The findings also implicate proteases as ligands to members of this orphan receptor family while providing new insights into how cysteine proteases contribute to itch. PMID:26216096

  14. Purification and characterization of a serine protease from Cucumis trigonus Roxburghi.

    PubMed

    Asif-Ullah, Mufti; Kim, Key-Sun; Yu, Yeon Gyu

    2006-05-01

    Kachri fruit, Cucumis trigonus Roxburghi, contains high protease activity and has been used as meat tenderizer in the Indian subcontinent. A 67 kDa serine protease from Kachri fruit was purified by DEAE-Sepharose and CM-Sepharose chromatography, whose optimum activity was at pH 11 and 70 degrees C. Its activity was strongly inhibited by PMSF, but not by EDTA, pepstatin, or cysteine protease inhibitors. The substrate specificity of the purified protease towards synthetic peptides was comparable to cucumisin, the first characterized subtilisin class plant protease from the sarcocarp of melon fruit (Cucumis melo). These characteristics, along with the N-terminal amino acid sequence, indicated that the isolated protease from Cucumis trigonus Roxburghi is a cucumisin homologue, which belongs to the serine protease family. PMID:16603211

  15. Development of marine biotechnology as a resource for novel proteases and their role in modern biotechnology.

    PubMed

    Homaei, Ahmad; Lavajoo, Fatemeh; Sariri, Reyhaneh

    2016-07-01

    Marine environment consists of the largest sources diversified genetic pool of material with an enormous potential for a wide variety of enzymes including proteases. A protease hydrolyzes the peptide bond and most of proteases possess many industrial applications. Marine proteases differ considerably from those found in internal or external organs of invertebrates and vertebrates. In common with all enzymes, external factors such as temperature, pH and type of media are important for the activity, catalytic efficiency, stability and proper functioning of proteases. In this review valuable characteristics of proteases in marine organisms and their applications are gathered from a wide literature survey. Considering their biochemical significance and their increasing importance in biotechnology, a thorough understanding of marine proteases functioning could be of prime importance.

  16. Preparation of Pt supported on WO 3-C with enhanced catalytic activity by microwave-pyrolysis method

    NASA Astrophysics Data System (ADS)

    Ye, Jilei; Liu, Jianguo; Zou, Zhigang; Gu, Jun; Yu, Tao

    The WO 3-C hybrid materials are prepared by intermittently microwave-pyrolysis using ammonium tungstate as the precursor, and then Pt nano-particles are deposited by microwave-assited polyol process on WO 3-C. The TEM images show the dispersion of ∼10 nm WO 3 particles size supported on carbon and ∼3 nm Pt metal crystallites supported on WO 3-C. XRD results illustrate that WO 3 presented as monoclinic phase and the content of WO 3 in WO 3/C and Pt/WO 3-C catalysts is further characterized by EDAX. Furthermore, XPS characterizations indicate that the interaction between Pt and WO 3 is dramatically enhanced after heat treatment at 200 °C. The activities of Pt/WO 3-C for the electrochemical oxidation of methanol are compared with Pt/C in acid solution by cyclic voltammetry, CO-stripping and chronoaperometry. Pt/WO 3-C catalyst calcined at 200 °C exhibits the highest activity per electrochemical active surface area for methanol oxidation and is 60 mV more negative for CO electro-oxidation than that of Pt/C and Pt/WO 3-C without heat treatment. The great enhancement of electrochemical performance may be due to the improvement of the synergistic effect between Pt and WO 3 in Pt/WO 3-C catalyst after heat treatment.

  17. ROSAT observations of the supernova remnant 3C 400.2

    NASA Astrophysics Data System (ADS)

    Saken, Jon M.; Long, K. S.; Blair, W. P.; Winkler, P. F.

    1995-04-01

    We have used the ROSAT point source proportional counter (PSPC) to examine the X-ray emission from 3C 400.2, a supernova remnant (SNR) which is a member of a class of remnants with limb-brightened radio and centrally condensed X-ray morphologies. The X-ray emission fills the radio shell and is characterized by an interior peak in the northwest region of the remnant. Otherwise, the surface brightness has a relatively smooth distribution. The X-ray peak is not correlated with any radio features or with the observed optical filaments. The PSPC X-ray spectrum is not well fitted by a power-law model but can be described in terms of thermal emission from a hot plasma with solar abundances. The only point source along the line of sight to the SNR is associated with a bright foreground F8 star. Thus the X-ray emission from 3C 400.2 is unlikely to be due to synchrotron radiation from an active pulsar. If the emission arises from a thermal plasma and the absorbing column along the line of sight to 3C 400.2 is 7.8 x 1021sq cm, then the temperature of the plasma is 0.27 keV, and the 0.4-2.4 keV X-ray luminosity is 1.3 x 1036 ergs/s for an assumed distance of 6 kpc. An X-ray hardness ratio map shows a slight increase in the hardness of the emission in the regions of the remnant with a higher X-ray surface brightness. Assuming uniform absorption across the remnant, this increase implies the temperature is approximately 1.5 times greater in the high surface brightness regions of SNR. The relatively uniform spectrum and the anticorrelation between X-ray and radio features seems to rule out the possibility that 3C 400.2 is actually two overlapping or interacting SNRs. The morphology of 3C 400.2 can be explained in terms of a multiphase interstellar medium (ISM) in which the primary shock is expanding into an ISM studded with dense cloudlets, if the clouds are evaporated or disrupted on a timescale which is long compared to the age of the SNR. It may also be possible to explain the

  18. ROSAT observations of the supernova remnant 3C 400.2

    NASA Technical Reports Server (NTRS)

    Saken, Jon M.; Long, K. S.; Blair, W. P.; Winkler, P. F.

    1995-01-01

    We have used the ROSAT point source proportional counter (PSPC) to examine the X-ray emission from 3C 400.2, a supernova remnant (SNR) which is a member of a class of remnants with limb-brightened radio and centrally condensed X-ray morphologies. The X-ray emission fills the radio shell and is characterized by an interior peak in the northwest region of the remnant. Otherwise, the surface brightness has a relatively smooth distribution. The X-ray peak is not correlated with any radio features or with the observed optical filaments. The PSPC X-ray spectrum is not well fitted by a power-law model but can be described in terms of thermal emission from a hot plasma with solar abundances. The only point source along the line of sight to the SNR is associated with a bright foreground F8 star. Thus the X-ray emission from 3C 400.2 is unlikely to be due to synchrotron radiation from an active pulsar. If the emission arises from a thermal plasma and the absorbing column along the line of sight to 3C 400.2 is 7.8 x 10(exp 21)sq cm, then the temperature of the plasma is 0.27 keV, and the 0.4-2.4 keV X-ray luminosity is 1.3 x 10(exp 36) ergs/s for an assumed distance of 6 kpc. An X-ray hardness ratio map shows a slight increase in the hardness of the emission in the regions of the remnant with a higher X-ray surface brightness. Assuming uniform absorption across the remnant, this increase implies the temperature is approximately 1.5 times greater in the high surface brightness regions of SNR. The relatively uniform spectrum and the anticorrelation between X-ray and radio features seems to rule out the possibility that 3C 400.2 is actually two overlapping or interacting SNRs. The morphology of 3C 400.2 can be explained in terms of a multiphase interstellar medium (ISM) in which the primary shock is expanding into an ISM studded with dense cloudlets, if the clouds are evaporated or disrupted on a timescale which is long compared to the age of the SNR. It may also be possible to

  19. Preliminary validation of the refractivity from the new radio occultation sounder GNOS/FY-3C

    NASA Astrophysics Data System (ADS)

    Liao, Mi; Zhang, Peng; Yang, Guang-Lin; Bi, Yan-Meng; Liu, Yan; Bai, Wei-Hua; Meng, Xiang-Guang; Du, Qi-Fei; Sun, Yue-Qiang

    2016-03-01

    As a new member of the space-based radio occultation sounders, the GNOS (Global Navigation Satellite System Occultation Sounder) mounted on Fengyun-3C (FY-3C) has been carrying out atmospheric sounding since 23 September 2013. GNOS takes approximately 800 daily measurements using GPS (Global Positioning System) and Chinese BDS (BeiDou navigation satellite) signals. In this work, the atmospheric refractivity profiles from GNOS were compared with the ones obtained from the co-located ECMWF (European Centre for Medium-Range Weather Forecasts) reanalysis. The mean bias of the refractivity obtained through GNOS GPS (BDS) was found to be approximately -0.09 % (-0.04 %) from the near surface to up to 46 km. While the average standard deviation was approximately 1.81 % (1.26 %), it was as low as 0.75 % (0.53 %) in the range of 5-25 km, where best sounding results are usually achieved. Further, COSMIC (Constellation Observing System for Meteorology, Ionosphere and Climate) and MetOp/ GRAS (GNSS Receiver for Atmospheric Sounding) radio occultation data were compared with the ECMWF reanalysis; the results thus obtained could be used as reference data for GNOS. Our results showed that GNOS/FY-3C meets the design requirements in terms of accuracy and precision of the sounder. It possesses a sounding capability similar to COSMIC and MetOp/GRAS in the vertical range of 0-30 km, though it needs further improvement above 30 km. Overall, it provides a new data source for the global numerical weather prediction (NWP) community.

  20. The Remarkable X-ray Spectrum of the Broad-Line Radio Galaxy 3C 445

    NASA Technical Reports Server (NTRS)

    Sambruna, R. M.; Reeves, J. N.; Braito, V.

    2007-01-01

    The nearby (z=0.057) radio-loud source 3C 445, optically classified as a Broad-Line Radio Galaxy, exhibits an X-ray spectrum strongly reminiscent of an obscured AGN, suggesting we are seeing this source at a relatively large angle from the radio jet. Here we present an archival 15 ks XMM-Newton observation of 3C 445 which confirms the remarkable complexity of its X-ray emission. The X-ray emission is described by a power law continuum with GAMMA approximately equal to 1.4, absorbed by several layers of cold gas, plus strong cold reflection. A narrow, unresolved Fe Kalpha emission line is detected, confirming previous findings, with EW approximately equal to 400 eV. A soft excess is present below 2 keV over the extrapolation of the hard X-ray power law, which we model with a power law with the same photon index and absorbed by a column density N(sub H)5 approximately equal to 10(sup 20) cm(sup -2) in excess to Galactic. Remarkably, a host of emission lines are present below 2 keV, confirming previous indications from ASCA, due to H- and He-like O, Mg, and Si. The detection of two features at 0.74 and 0.87 keV, identified with OVII and OVIII Radiative Recombination Continuum features, suggest an origin of the lines from a photoionized gas, with properties very similar to radio-quiet obscured AGN. Two different ionized media, or a single stratified medium, are required to fit the soft X-ray data satisfactorily. The similarity of the X-ray spectrum of 3C 445 to Seyferts underscores that the central engines of radio-loud and radio-quiet AGN similarly host both cold and warm gas.