Sample records for nia protease recombinant

  1. Characterization of active-site residues of the NIa protease from tobacco vein mottling virus. (United States)

    Hwang, D C; Kim, D H; Lee, J S; Kang, B H; Han, J; Kim, W; Song, B D; Choi, K Y


    Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased kcat marginally by about 4.7-fold and increased Km by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in kcat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pKa values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.

  2. The nuclear inclusion a (NIa protease of turnip mosaic virus (TuMV cleaves amyloid-β.

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    Hye-Eun Han

    Full Text Available BACKGROUND: The nuclear inclusion a (NIa protease of turnip mosaic virus (TuMV is responsible for the processing of the viral polyprotein into functional proteins. NIa was previously shown to possess a relatively strict substrate specificity with a preference for Val-Xaa-His-Gln↓, with the scissile bond located after Gln. The presence of the same consensus sequence, Val(12-His-His-Gln(15, near the presumptive α-secretase cleavage site of the amyloid-β (Aβ peptide led us to hypothesize that NIa could possess activity against Aβ. METHODOLOGY/PRINCIPAL FINDINGS: Western blotting results showed that oligomeric as well as monomeric forms of Aβ can be degraded by NIa in vitro. The specific cleavage of Aβ was further confirmed by mass spectrometry analysis. NIa was shown to exist predominantly in the cytoplasm as observed by immunofluorescence microscopy. The overexpression of NIa in B103 neuroblastoma cells resulted in a significant reduction in cell death caused by both intracellularly generated and exogenously added Aβ. Moreover, lentiviral-mediated expression of NIa in APP(sw/PS1 transgenic mice significantly reduced the levels of Aβ and plaques in the brain. CONCLUSIONS/SIGNIFICANCE: These results indicate that the degradation of Aβ in the cytoplasm could be a novel strategy to control the levels of Aβ, plaque formation, and the associated cell death.

  3. A dual protease approach for expression and affinity purification of recombinant proteins. (United States)

    Raran-Kurussi, Sreejith; Waugh, David S


    We describe a new method for affinity purification of recombinant proteins using a dual protease protocol. Escherichia coli maltose binding protein (MBP) is employed as an N-terminal tag to increase the yield and solubility of its fusion partners. The MBP moiety is then removed by rhinovirus 3C protease, prior to purification, to yield an N-terminally His6-tagged protein. Proteins that are only temporarily rendered soluble by fusing them to MBP are readily identified at this stage because they will precipitate after the MBP tag is removed by 3C protease. The remaining soluble His6-tagged protein, if any, is subsequently purified by immobilized metal affinity chromatography (IMAC). Finally, the N-terminal His6 tag is removed by His6-tagged tobacco etch virus (TEV) protease to yield the native recombinant protein, and the His6-tagged contaminants are removed by adsorption during a second round of IMAC, leaving only the untagged recombinant protein in the column effluent. The generic strategy described here saves time and effort by removing insoluble aggregates at an early stage in the process while also reducing the tendency of MBP to "stick" to its fusion partners during affinity purification. Published by Elsevier Inc.

  4. Companion Protease Inhibitors for the In Situ Protection of Recombinant Proteins in Plants. (United States)

    Robert, Stéphanie; Jutras, Philippe V; Khalf, Moustafa; D'Aoust, Marc-André; Goulet, Marie-Claire; Sainsbury, Frank; Michaud, Dominique


    We previously described a procedure for the use of plant protease inhibitors as "companion" accessory proteins to prevent unwanted proteolysis of clinically useful recombinant proteins in leaf crude protein extracts (Benchabane et al. Methods Mol Biol 483:265-273, 2009). Here we describe the use of these inhibitors for the protection of recombinant proteins in planta, before their extraction from leaf tissues. A procedure is first described involving inhibitors co-expressed along-and co-migrating-with the protein of interest in host plant cells. An alternative, single transgene scheme is then described involving translational fusions of the recombinant protein and companion inhibitor. These approaches may allow for a significant improvement of protein steady-state levels in leaves, comparable to yield improvements observed with protease-deficient strains of less complex protein expression hosts such as E. coli or yeasts.

  5. Recombinant expression and functional analysis of proteases from Streptococcus pneumoniae, Bacillus anthracis, and Yersinia pestis

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    Pieper Rembert


    Full Text Available Abstract Background Uncharacterized proteases naturally expressed by bacterial pathogens represents important topic in infectious disease research, because these enzymes may have critical roles in pathogenicity and cell physiology. It has been observed that cloning, expression and purification of proteases often fail due to their catalytic functions which, in turn, cause toxicity in the E. coli heterologous host. Results In order to address this problem systematically, a modified pipeline of our high-throughput protein expression and purification platform was developed. This included the use of a specific E. coli strain, BL21(DE3 pLysS to tightly control the expression of recombinant proteins and various expression vectors encoding fusion proteins to enhance recombinant protein solubility. Proteases fused to large fusion protein domains, maltosebinding protein (MBP, SP-MBP which contains signal peptide at the N-terminus of MBP, disulfide oxidoreductase (DsbA and Glutathione S-transferase (GST improved expression and solubility of proteases. Overall, 86.1% of selected protease genes including hypothetical proteins were expressed and purified using a combination of five different expression vectors. To detect novel proteolytic activities, zymography and fluorescence-based assays were performed and the protease activities of more than 46% of purified proteases and 40% of hypothetical proteins that were predicted to be proteases were confirmed. Conclusions Multiple expression vectors, employing distinct fusion tags in a high throughput pipeline increased overall success rates in expression, solubility and purification of proteases. The combinatorial functional analysis of the purified proteases using fluorescence assays and zymography confirmed their function.

  6. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses (United States)


    Background Baculovirus comprise the largest group of insect viruses most studied worldwide, mainly because they efficiently kill agricutural insect pests. In this study, two recombinant baculoviruses containing the ScathL gene from Sarcophaga peregrina (vSynScathL), and the Keratinase gene from the fungus Aspergillus fumigatus (vSynKerat), were constructed. and their insecticidal properties analysed against Spodoptera frugiperda larvae. Results Bioassays of third-instar and neonate S. frugiperda larvae with vSynScathL and vSynKerat showed a decrease in the time needed to kill the infected insects when compared to the wild type virus. We have also shown that both recombinants were able to increase phenoloxidase activity in the hemolymph of S. frugiperda larvae. The expression of proteases in infected larvae resulted in destruction of internal tissues late in infection, which could be the reason for the increased viral speed of kill. Conclusions Baculoviruses and their recombinant forms constitute viable alternatives to chemical insecticides. Recombinant baculoviruses containing protease genes can be added to the list of engineered baculoviruses with great potential to be used in integrated pest management programs. PMID:20587066

  7. Serological Analysis of Immunogenic Properties of Recombinant Meningococcus IgA1 Protease-Based Proteins. (United States)

    Kotelnikova, O V; Zinchenko, A A; Vikhrov, A A; Alliluev, A P; Serova, O V; Gordeeva, E A; Zhigis, L S; Zueva, V S; Razgulyaeva, O A; Melikhova, T D; Nokel, E A; Drozhzhina, E Yu; Rumsh, L D


    Using the genome sequence of IgA1 protease of N. meningitidis of serogroup B, four recombinant proteins of different structure and molecular weight were constructed. These proteins were equal in inducing the formation of specific antibodies to IgA1 protease and had protective properties against meningococci. In the sera of immunized mice, anti-IgA1 protease antibodies were detected by whole-cell ELISA, which indicated the presence of IgA1 protease on the surface of these bacteria. We hypothesized that the protective properties of IgA1 protease-based antigens and IgA1 protease analogs could be realized not only via impairment of bacterium adhesion to the mucosa, but also via suppression of this pathogen in the organism. The presented findings seem promising for using these proteins as the basis for anti-meningococcus vaccine.

  8. In vitro Ca(2+)-dependent maturation of milk-clotting recombinant Epr: minor extracellular protease: from Bacillus licheniformis. (United States)

    Ageitos, José Manuel; Vallejo, Juan Andrés; Serrat, Manuel; Sánchez-Pérez, Angeles; Villa, Tomás G


    The minor extracellular protease (Epr) is secreted into the culture medium during Bacillus licheniformis, strain USC13, stationary phase of growth. Whereas, B. subtilis Epr has been reported to be involved in swarming; the B. licheniformis protease is also involved in milk-clotting as shown by the curd forming ability of culture broths expressing this protein. The objectives of this study are the characterization of recombinant B. licheniformis Epr (minor extracellular protease) and the determination of its calcium-dependent activation process. In this work, we have cloned and expressed B. licheniformis Epr in Escherichia coli. We were also able to construct a tridimensional model for Epr based on its homology to Thermococcus kodakarensis pro-tk-subtilisin 2e1p, fervidolysin from Fervidobacterium pennivorans 1rv6, and B. lentus 1GCI subtilisin. Recombinant Epr was accumulated into inclusion bodies; after protein renaturation, Epr undergoes an in vitro calcium-dependent activation, similar to that described for tk protease. The recombinant Epr is capable of producing milk curds with the same clotting activity previously described for the native B. licheniformis Epr enzyme although further rheological and industrial studies should be carried out to confirm its real applicability. This work represents for the first time that Epr may be successfully expressed in a non-bacilli microorganism.

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

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    Raheem Ullah

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

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

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


    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.

  11. A recombinant fusion protein-based, fluorescent protease assay for high throughput-compatible substrate screening. (United States)

    Bozóki, Beáta; Gazda, Lívia; Tóth, Ferenc; Miczi, Márió; Mótyán, János András; Tőzsér, József


    In connection with the intensive investigation of proteases, several methods have been developed for analysis of the substrate specificity. Due to the great number of proteases and the expected target molecules to be analyzed, time- and cost-efficient high-throughput screening (HTS) methods are preferred. Here we describe the development and application of a separation-based HTS-compatible fluorescent protease assay, which is based on the use of recombinant fusion proteins as substrates of proteases. The protein substrates used in this assay consists of N-terminal (hexahistidine and maltose binding protein) fusion tags, cleavage sequences of the tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein (mApple or mTurquoise2). The assay is based on the fluorimetric detection of the fluorescent proteins, which are released from the magnetic bead-attached substrates by the proteolytic cleavage. The protease assay has been applied for activity measurements of TEV and HIV-1 proteases to test the suitability of the system for enzyme kinetic measurements, inhibition studies, and determination of pH optimum. We also found that denatured fluorescent proteins can be renatured after SDS-PAGE of denaturing conditions, but showed differences in their renaturation abilities. After in-gel renaturation both substrates and cleavage products can be identified by in-gel UV detection. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Differential sensitivity of 5'UTR-NS5A recombinants of hepatitis C virus genotypes 1-6 to protease and NS5A inhibitors

    DEFF Research Database (Denmark)

    Li, Yi-Ping; Ramirez, Santseharay; Humes, Daryl


    BACKGROUND & AIMS: Hepatitis C virus (HCV) therapy will benefit from the preclinical evaluation of direct-acting antiviral (DAA) agents in infectious culture systems that test the effects on different virus genotypes. We developed HCV recombinants comprising the 5' untranslated region-NS5A (5-5A...... daclatasvir. The 1a(TN) 5-5A and JFH1-independent full-length viruses had similar levels of sensitivity to the DAA agents, validating the 5-5A recombinants as surrogates for full-length viruses in DAA testing. Compared with the 1a(TN) full-length virus, the 3a(S52) 5-5A recombinant was highly resistant to all...... protease inhibitors, and the 4a(ED43) recombinant was highly resistant to telaprevir and boceprevir, but most sensitive to other protease inhibitors. Compared with other protease inhibitors, MK-5172 had exceptional potency against all HCV genotypes. The NS5A inhibitor daclatasvir had the highest potency...

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

    International Nuclear Information System (INIS)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  15. Glycosylation analysis of recombinant neutral protease I from Aspergillus oryzae expressed in Pichia pastoris. (United States)

    Lei, Da; Xu, Yang; He, Qinghua; Pang, Yifeng; Chen, Bo; Xiong, Liang; Li, Yanping


    Neutral protease I from Aspergillus oryzae 3.042 was expressed in Pichia pastoris and its N-glycosylation properties were analyzed. After purification by nickel-affinity chromatography column, the recombinant neutral protease (rNPI) was confirmed to be N-glycosylated by periodicacid/Schiff's base staining and Endo H digestion. Moreover, the deglycosylated protein's molecular weight decreased to 43.3 kDa from 54.5 kDa analyzed by SDS-PAGE and MALDI-TOF-MS, and the hyperglycosylation extent was 21 %. The N-glycosylation site of rNPI was analyzed by nano LC-MS/MS after digesting by trypsin and Glu-C, and the unique potential site Asn41 of mature peptide was found to be glycosylated. Homology modeling of the 3D structure of rNPI indicated that the attached N-glycans hardly affected neutral protease's activity due to the great distance away from the active site of the enzyme.

  16. Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells. (United States)

    Mandal, Manoj K; Fischer, Rainer; Schillberg, Stefan; Schiermeyer, Andreas


    Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease-deficient tobacco BY-2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY-2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full-length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV-1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four-fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N-terminal sequencing data revealed that the antibody has two cleavage sites within the CDR-H3 and one site at the end of the H4-framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures. Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

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    Kishor Duwadi

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

  18. PhAP protease from Pseudoalteromonas haloplanktis TAC125: Gene cloning, recombinant production in E. coli and enzyme characterization (United States)

    de Pascale, D.; Giuliani, M.; De Santi, C.; Bergamasco, N.; Amoresano, A.; Carpentieri, A.; Parrilli, E.; Tutino, M. L.


    Cold-adapted proteases have been found to be the dominant activity throughout the cold marine environment, indicating their importance in bacterial acquisition of nitrogen-rich complex organic compounds. However, few extracellular proteases from marine organisms have been characterized so far, and the mechanisms that enable their activity in situ are still largely unknown. Aside from their ecological importance and use as model enzyme for structure/function investigations, cold-active proteolytic enzymes offer great potential for biotechnological applications. Our studies on cold adapted proteases were performed on exo-enzyme produced by the Antarctic marine bacterium Pseudoalteromonas haloplanktis TAC125. By applying a proteomic approach, we identified several proteolytic activities from its culture supernatant. PhAP protease was selected for further investigations. The encoding gene was cloned and the protein was recombinantly produced in E. coli cells. The homogeneous product was biochemically characterised and it turned out that the enzyme is a Zn-dependent aminopeptidase, with an activity dependence from assay temperature typical of psychrophilic enzymes.

  19. Novel ATPase activity of the polyprotein intermediate, Viral Protein genome-linked-Nuclear Inclusion-a protease, of Pepper vein banding potyvirus

    International Nuclear Information System (INIS)

    Mathur, Chhavi; Savithri, Handanahal S.


    Highlights: ► Pepper vein banding potyvirus VPg harbors Walker motifs. ► VPg exhibits ATPase activity in the presence of NIa-Pro. ► Plausible structural and functional interplay between VPg and NIa-Pro. ► Functional relevance of prolonged presence of VPg-Pro during infection. -- Abstract: Potyviruses temporally regulate their protein function by polyprotein processing. Previous studies have shown that VPg (Viral Protein genome-linked) of Pepper vein banding virus interacts with the NIa-Pro (Nuclear Inclusion-a protease) domain, and modulates the kinetics of the protease. In the present study, we report for the first time that VPg harbors the Walker motifs A and B, and the presence of NIa-Pro, especially in cis (cleavage site (E191A) VPg-Pro mutant), is essential for manifestation of the ATPase activity. Mutation of Lys47 (Walker motif A) and Asp88:Glu89 (Walker motif B) to alanine in E191A VPg-Pro lead to reduced ATPase activity, confirming that this activity was inherent to VPg. We propose that potyviral VPg, established as an intrinsically disordered domain, undergoes plausible structural alterations upon interaction with globular NIa-Pro which induces the ATPase activity.

  20. Efficient sortase-mediated N-terminal labeling of TEV protease cleaved recombinant proteins. (United States)

    Sarpong, Kwabena; Bose, Ron


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

  1. The Coat Protein and NIa Protease of Two Potyviridae Family Members Independently Confer Superinfection Exclusion (United States)

    French, Roy


    ABSTRACT Superinfection exclusion (SIE) is an antagonistic virus-virus interaction whereby initial infection by one virus prevents subsequent infection by closely related viruses. Although SIE has been described in diverse viruses infecting plants, humans, and animals, its mechanisms, including involvement of specific viral determinants, are just beginning to be elucidated. In this study, SIE determinants encoded by two economically important wheat viruses, Wheat streak mosaic virus (WSMV; genus Tritimovirus, family Potyviridae) and Triticum mosaic virus (TriMV; genus Poacevirus, family Potyviridae), were identified in gain-of-function experiments that used heterologous viruses to express individual virus-encoded proteins in wheat. Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg, or NIb cistrons permitted efficient superinfection by WSMV expressing green fluorescent protein (WSMV-GFP). In contrast, wheat infected with TriMV expressing WSMV NIa-Pro or coat protein (CP) substantially excluded superinfection by WSMV-GFP, suggesting that both of these cistrons are SIE effectors encoded by WSMV. Importantly, SIE is due to functional WSMV NIa-Pro or CP rather than their encoding RNAs, as altering the coded protein products by minimally changing RNA sequences led to abolishment of SIE. Deletion mutagenesis further revealed that elicitation of SIE by NIa-Pro requires the entire protein while CP requires only a 200-amino-acid (aa) middle fragment (aa 101 to 300) of the 349 aa. Strikingly, reciprocal experiments with WSMV-mediated expression of TriMV proteins showed that TriMV CP, and TriMV NIa-Pro to a lesser extent, likewise excluded superinfection by TriMV-GFP. Collectively, these data demonstrate that WSMV- and TriMV-encoded CP and NIa-Pro proteins are effectors of SIE and that these two proteins trigger SIE independently of each other. IMPORTANCE Superinfection exclusion (SIE) is an antagonistic virus-virus interaction that

  2. The putative serine protease inhibitor Api m 6 from Apis mellifera venom: recombinant and structural evaluation. (United States)

    Michel, Y; McIntyre, M; Ginglinger, H; Ollert, M; Cifuentes, L; Blank, S; Spillner, E


    Immunoglobulin (Ig) E-mediated reactions to honeybee venom can cause severe anaphylaxis, sometimes with fatal consequences. Detailed knowledge of the allergic potential of all venom components is necessary to ensure proper diagnosis and treatment of allergy and to gain a better understanding of the allergological mechanisms of insect venoms. Our objective was to undertake an immunochemical and structural evaluation of the putative low-molecular-weight serine protease inhibitor Api m 6, a component of honeybee venom. We recombinantly produced Api m 6 as a soluble protein in Escherichia coli and in Spodoptera frugiperda (Sf9) insect cells.We also assessed specific IgE reactivity of venom-sensitized patients with 2 prokaryotically produced Api m 6 variants using enzyme-linked immunosorbent assay. Moreover, we built a structural model ofApi m 6 and compared it with other protease inhibitor structures to gain insights into the function of Api m 6. In a population of 31 honeybee venom-allergic patients, 26% showed specific IgE reactivity with prokaryotically produced Api m 6, showing it to be a minor but relevant allergen. Molecular modeling of Api m 6 revealed a typical fold of canonical protease inhibitors, supporting the putative function of this venom allergen. Although Api m 6 has a highly variant surface charge, its epitope distribution appears to be similar to that of related proteins. Api m 6 is a honeybee venom component with IgE-sensitizing potential in a fraction of venom-allergic patients. Recombinant Api m 6 can help elucidate individual component-resolved reactivity profiles and increase our understanding of immune responses to low-molecular-weight allergens

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

    African Journals Online (AJOL)



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

  4. High throughput in vivo protease inhibitor selection platform

    DEFF Research Database (Denmark)


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

  5. Optimization of Soluble Expression and Purification of Recombinant Human Rhinovirus Type-14 3C Protease Using Statistically Designed Experiments: Isolation and Characterization of the Enzyme. (United States)

    Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos


    Human rhinovirus (HRV) 3C protease is widely used in recombinant protein production for various applications such as biochemical characterization and structural biology projects to separate recombinant fusion proteins from their affinity tags in order to prevent interference between these tags and the target proteins. Herein, we report the optimization of expression and purification conditions of glutathione S-transferase (GST)-tagged HRV 3C protease by statistically designed experiments. Soluble expression of GST-HRV 3C protease was initially optimized by response surface methodology (RSM), and a 5.5-fold increase in enzyme yield was achieved. Subsequently, we developed a new incomplete factorial (IF) design that examines four variables (bacterial strain, expression temperature, induction time, and inducer concentration) in a single experiment. The new design called Incomplete Factorial-Strain/Temperature/Time/Inducer (IF-STTI) was validated using three GST-tagged proteins. In all cases, IF-STTI resulted in only 10% lower expression yields than those obtained by RSM. Purification of GST-HRV 3C was optimized by an IF design that examines simultaneously the effect of the amount of resin, incubation time of cell lysate with resin, and glycerol and DTT concentration in buffers, and a further 15% increase in protease recovery was achieved. Purified GST-HRV 3C protease was active at both 4 and 25 °C in a variety of buffers.

  6. Construction and application of recombinant strain for the production of an alkaline protease from Bacillus licheniformis. (United States)

    Lin, Songyi; Zhang, Meishuo; Liu, Jingbo; Jones, Gregory S


    The alkaline protease gene, Apr, from Bacillus licheniformis 2709 was cloned into an expression vector pET - 28b (+), to yield the recombinant plasmid pET-28b (+) - Apr. The pET-28b (+) - Apr was expressed in a high expression strain E. coli BL21. The amino acid sequence deduced from the DNA sequence analysis revealed a 98% identity to that of Bacillus licheniformis 2709. Sodium salt-Polyacrylamide gel electrophoresis (SDS-PAGE) was used to access the protein expression. SDS-PAGE analysis indicated a protein of Mr of 38.8 kDa. The medium components and condition of incubation were optimized for the growth state of a recombinant strain. The optimal composition of production medium was composed of glucose 8 g/L, peptone 8 g/L and salt solution 10 mL. The samples were incubated on a rotary shaker of 180 r/min at 37°C for 24 h. Copyright © 2014. Published by Elsevier B.V.

  7. Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients. (United States)

    Gehringer, Heike; Von der Helm, Klaus; Seelmeir, Sigrid; Weissbrich, Benedikt; Eberle, Josef; Nitschko, Hans


    A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

  8. Adapted J6/JFH1-based Hepatitis C virus recombinants with genotype-specific NS4A show similar efficacies against lead protease inhibitors, alpha interferon, and a putative NS4A inhibitor

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Sanne B; Serre, Stéphanie B N


    To facilitate studies of hepatitis C virus (HCV) NS4A, we aimed at developing J6/JFH1-based recombinants with genotype 1- to 7-specific NS4A proteins. We developed efficient culture systems expressing NS4A proteins of genotypes (isolates) 1a (H77 and TN), 1b (J4), 2a (J6), 4a (ED43), 5a (SA13), 6a...... (HK6a), and 7a (QC69), with peak infectivity titers of ∼3.5 to 4.5 log10 focus-forming units per ml. Except for genotype 2a (J6), growth depended on adaptive mutations identified in long-term culture. Genotype 1a, 1b, and 4a recombinants were adapted by amino acid substitutions F772S (p7) and V1663A...... (NS4A), while 5a, 6a, and 7a recombinants required additional substitutions in the NS3 protease and/or NS4A. We demonstrated applicability of the developed recombinants for study of antivirals. Genotype 1 to 7 NS4A recombinants showed similar responses to the protease inhibitors telaprevir (VX-950...

  9. Enhanced Abscisic Acid-Mediated Responses in nia1nia2noa1-2 Triple Mutant Impaired in NIA/NR- and AtNOA1-Dependent Nitric Oxide Biosynthesis in Arabidopsis1[W (United States)

    Lozano-Juste, Jorge; León, José


    Nitric oxide (NO) regulates a wide range of plant processes from development to environmental adaptation. Despite its reported regulatory functions, it remains unclear how NO is synthesized in plants. We have generated a triple nia1nia2noa1-2 mutant that is impaired in nitrate reductase (NIA/NR)- and Nitric Oxide-Associated1 (AtNOA1)-mediated NO biosynthetic pathways. NO content in roots of nia1nia2 and noa1-2 plants was lower than in wild-type plants and below the detection limit in nia1nia2noa1-2 plants. NIA/NR- and AtNOA1-mediated biosynthesis of NO were thus active and responsible for most of the NO production in Arabidopsis (Arabidopsis thaliana). The nia1nia2noa1-2 plants displayed reduced size, fertility, and seed germination potential but increased dormancy and resistance to water deficit. The increasing deficiency in NO of nia1nia2, noa1-2, and nia1nia2noa1-2 plants correlated with increased seed dormancy, hypersensitivity to abscisic acid (ABA) in seed germination and establishment, as well as dehydration resistance. In nia1nia2noa1-2 plants, enhanced drought tolerance was due to a very efficient stomata closure and inhibition of opening by ABA, thus uncoupling NO from ABA-triggered responses in NO-deficient guard cells. The NO-deficient mutants in NIA/NR- and AtNOA1-mediated pathways in combination with the triple mutant will be useful tools to functionally characterize the role of NO and the contribution of both biosynthetic pathways in regulating plant development and defense. PMID:20007448

  10. Loss of nitrate reductases NIA1 and NIA2 impairs stomatal closure by altering genes of core ABA signaling components in Arabidopsis. (United States)

    Zhao, Chenchen; Cai, Shengguan; Wang, Yizhou; Chen, Zhong-Hua


    Nitrate reductases NIA1 and NIA2 determine NO production in plants and are critical to abscisic acid (ABA)-induced stomatal closure. However, the role for NIA1 and NIA2 in ABA signaling has not been paid much attention in nitrate reductase loss-of-function mutant nia1nia2. Recently, we have demonstrated that ABA-inhibited K(+)in current and ABA-enhanced slow anion current were absent in nia1nia2. Exogenous NO restored regulation of these channels for stomatal closure in nia1nia2. In this study, we found that mutating NIA1 and NIA2 impaired nearly all the key components of guard cell ABA signaling pathway in Arabidopsis. We also propose a simplified model for ABA signaling in the nia1nia2 mutant.

  11. NIA Aging Cell Repository (United States)

    Federal Laboratory Consortium — To facilitate aging research on cells in culture, the NIA provides support for the NIA Aging Cell Repository, located at the Coriell Institute for Medical Research...

  12. SjAPI, the first functionally characterized Ascaris-type protease inhibitor from animal venoms.

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    Zongyun Chen

    Full Text Available BACKGROUND: Serine protease inhibitors act as modulators of serine proteases, playing important roles in protecting animal toxin peptides from degradation. However, all known serine protease inhibitors discovered thus far from animal venom belong to the Kunitz-type subfamily, and whether there are other novel types of protease inhibitors in animal venom remains unclear. PRINCIPAL FINDINGS: Here, by screening scorpion venom gland cDNA libraries, we identified the first Ascaris-type animal toxin family, which contains four members: Scorpiops jendeki Ascaris-type protease inhibitor (SjAPI, Scorpiops jendeki Ascaris-type protease inhibitor 2 (SjAPI-2, Chaerilus tricostatus Ascaris-type protease inhibitor (CtAPI, and Buthus martensii Ascaris-type protease inhibitor (BmAPI. The detailed characterization of Ascaris-type peptide SjAPI from the venom gland of scorpion Scorpiops jendeki was carried out. The mature peptide of SjAPI contains 64 residues and possesses a classical Ascaris-type cysteine framework reticulated by five disulfide bridges, different from all known protease inhibitors from venomous animals. Enzyme and inhibitor reaction kinetics experiments showed that recombinant SjAPI was a dual function peptide with α-chymotrypsin- and elastase-inhibiting properties. Recombinant SjAPI inhibited α-chymotrypsin with a Ki of 97.1 nM and elastase with a Ki of 3.7 μM, respectively. Bioinformatics analyses and chimera experiments indicated that SjAPI contained the unique short side chain functional residues "AAV" and might be a useful template to produce new serine protease inhibitors. CONCLUSIONS/SIGNIFICANCE: To our knowledge, SjAPI is the first functionally characterized animal toxin peptide with an Ascaris-type fold. The structural and functional diversity of animal toxins with protease-inhibiting properties suggested that bioactive peptides from animal venom glands might be a new source of protease inhibitors, which will accelerate the

  13. Improving the performance of industrial ethanol-producing yeast by expressing the aspartyl protease on the cell surface. (United States)

    Guo, Zhong-peng; Zhang, Liang; Ding, Zhong-yang; Wang, Zheng-Xiang; Shi, Gui-Yang


    The yeasts used in fuel ethanol manufacture are unable to metabolize soluble proteins. The PEP4 gene, encoding a vacuolar aspartyl protease in Saccharomyces cerevisiae, was either secretively or cell-surface anchored expressed in industrial ethanol-producing S. cerevisiae. The obtained recombinant strains APA (expressing the protease secretively) and APB (expressing the protease on the cell wall) were studied under ethanol fermentation conditions in feed barley cultures. The effects of expression of the protease on product formation, growth and cell protein content were measured. The biomass yield of the wild-type was clearly lower than that of the recombinant strains (0.578 ± 0.12 g biomass/g glucose for APA and 0.582 ± 0.08 g biomass/g glucose for APB). In addition, nearly 98-99% of the theoretical maximum level of ethanol yield was achieved (relative to the amount of substrate consumed) for the recombinant strains, while limiting the nitrogen source resulted in dissatisfactory fermentation for the wild-type and more than 30 g/l residual sugar was detected at the end of fermentation. In addition, higher growth rate, viability and lower yields of byproducts such as glycerol and pyruvic acid for recombinant strains were observed. Expressing acid protease can be expected to lead to a significant increase in ethanol productivity. Copyright © 2010 John Wiley & Sons, Ltd.

  14. An efficient procedure for the expression and purification of HIV-1 protease from inclusion bodies. (United States)

    Nguyen, Hong-Loan Thi; Nguyen, Thuy Thi; Vu, Quy Thi; Le, Hang Thi; Pham, Yen; Trinh, Phuong Le; Bui, Thuan Phuong; Phan, Tuan-Nghia


    Several studies have focused on HIV-1 protease for developing drugs for treating AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. However, large-scale expression and purification of this enzyme is difficult mainly because of its low expression and solubility. In this study, we constructed 9 recombinant plasmids containing a sequence encoding HIV-1 protease along with different fusion tags and examined the expression of the enzyme from these plasmids. Of the 9 plasmids, pET32a(+) plasmid containing the HIV-1 protease-encoding sequence along with sequences encoding an autocleavage site GTVSFNF at the N-terminus and TEV plus 6× His tag at the C-terminus showed the highest expression of the enzyme and was selected for further analysis. The recombinant protein was isolated from inclusion bodies by using 2 tandem Q- and Ni-Sepharose columns. SDS-PAGE of the obtained HIV-1 protease produced a single band of approximately 13 kDa. The enzyme was recovered efficiently (4 mg protein/L of cell culture) and had high specific activity of 1190 nmol min(-1) mg(-1) at an optimal pH of 4.7 and optimal temperature of 37 °C. This procedure for expressing and purifying HIV-1 protease is now being scaled up to produce the enzyme on a large scale for its application. Copyright © 2015 Elsevier Inc. All rights reserved.

  15. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis. (United States)

    Kadek, Alan; Tretyachenko, Vyacheslav; Mrazek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, Martial; Schriemer, David C; Man, Petr


    Carnivorous plants of the genus Nepenthes produce their own aspartic proteases, nepenthesins, to digest prey trapped in their pitchers. Nepenthesins differ significantly in sequence from other aspartic proteases in the animal or even plant kingdoms. This difference, which also brings more cysteine residues into the structure of these proteases, can be a cause of uniquely high temperature and pH stabilities of nepenthesins. Their detailed structure characterization, however, has not previously been possible due to low amounts of protease present in the pitcher fluid and also due to limited accessibility of Nepenthes plants. In the present study we describe a convenient way for obtaining high amounts of nepenthesin-1 from Nepenthes gracilis using heterologous production in Escherichia coli. The protein can be easily refolded in vitro and its characteristics are very close to those described for a natural enzyme isolated from the pitcher fluid. Similarly to the natural enzyme, recombinant nepenthesin-1 is sensitive to denaturing and reducing agents. It also has maximal activity around pH 2.5, shows unusual stability at high pH and its activity is not irreversibly inhibited even after prolonged incubation in the basic pH range. On the other hand, temperature stability of the recombinant enzyme is lower in comparison with the natural enzyme, which can be attributed to missing N-glycosylation in the recombinant protein. Copyright © 2013 Elsevier Inc. All rights reserved.

  16. Purification and characterization of naturally occurring HIV-1 (South African subtype C) protease mutants from inclusion bodies. (United States)

    Maseko, Sibusiso B; Natarajan, Satheesh; Sharma, Vikas; Bhattacharyya, Neelakshi; Govender, Thavendran; Sayed, Yasien; Maguire, Glenn E M; Lin, Johnson; Kruger, Hendrik G


    Human immunodeficiency virus (HIV) infections in sub-Saharan Africa represent about 56% of global infections. Many studies have targeted HIV-1 protease for the development of drugs against AIDS. Recombinant HIV-1 protease is used to screen new drugs from synthetic compounds or natural substances. Along with the wild type (C-SA) we also over-expressed and characterized two mutant forms from patients that had shown resistance to protease inhibitors. Using recombinant DNA technology, we constructed three recombinant plasmids in pGEX-6P-1 and expressed them containing a sequence encoding wild type HIV protease and two mutants (I36T↑T contains 100 amino acids and L38L↑N↑L contains 101 amino acids). These recombinant proteins were isolated from inclusion bodies by using QFF anion exchange and GST trap columns. In SDS-PAGE, we obtained these HIV proteases as single bands of approximately 11.5, 11.6 and 11.7 kDa for the wild type, I36T↑Tand L38L↑N↑L mutants, respectively. The enzyme was recovered efficiently (0.25 mg protein/L of Escherichia coli culture) and had high specific activity of 2.02, 2.20 and 1.33 μmol min(-1) mg(-1) at an optimal pH of 5 and temperature of 37 °C for the wild type, I36T↑T and L38L↑N↑L, respectively. The method employed here provides an easy and rapid purification of the HIV-1(C-SA) protease from the inclusion bodies, with high yield and high specific activities. Copyright © 2016 Elsevier Inc. All rights reserved.

  17. New tick records in Rondônia, Western Brazilian Amazon Novos relatos de carrapatos em Rondônia, Amazônia ocidental brasileira

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    Marcelo Bahia Labruna


    Full Text Available In the present study, we provide new tick records from Vilhena Municipality, in the Southeast of the State of Rondônia, Northern Brazil. Ticks collected from a capybara, Hydrochoerus hydrochaeris (Linnaeus, were identified as Amblyomma romitii Tonelli-Rondelli (1 female, and Amblyomma sp. (1 larva. Ticks collected from a harpy eagle, Harpia harpyja (Linnaeus, were identified as Amblyomma cajennense (Fabricius (16 nymphs and Haemaphysalis juxtakochi Cooley (1 nymph. Ticks collected from a yellow-footed tortoise, Chelonoidis denticulada (Linnaeus, were identified as Amblyomma rotundatum Koch (10 females, 2 nymphs, and Amblyomma sp. (2 larvae. The present record of A. romitii is the first in the State of Rondônia, and represents the southernmost record for this tick species, indicating that its distribution area is much larger than currently recognized. Although both A. cajennense and H. juxtakochi have been reported parasitizing various bird species, we provide the first tick records on a harpy eagle. A. rotundatum is widespread in the State of Rondônia, and has been previously reported on the yellow-footed tortoise. The present records increase the tick fauna of Rondônia to 26 species.O presente estudo relata novos achados de carrapatos provenientes do Município de Vilhena, Sudeste do Estado de Rondônia, na região Norte do Brasil. Carrapatos colhidos de uma capivara, Hydrochoerus hydrochaeris (Linnaeus, foram identificados como Amblyomma romitii Tonelli-Rondelli (1 fêmea e Amblyomma sp. (1 larva. Carrapatos colhidos de uma águia harpia, Harpia harpyja (Linnaeus, foram identificados como Amblyomma cajennense (Fabricius (16 ninfas e Haemaphysalis juxtakochi Cooley (1 ninfa. Carrapatos colhidos de um jabuti, Chelonoidis denticulada (Linnaeus, foram identificados como Amblyomma rotundatum Koch (10 fêmeas, 2 ninfas e Amblyomma sp. (2 larvas. O presente achado de A. romitii é o primeiro no Estado de Rondônia, representando o achado mais

  18. Superstructure of NiAs

    International Nuclear Information System (INIS)

    Nozue, Tatsuhiro; Kobayashi, Hisao; Kamimura, Takashi; Yamaguchi, Yasuo


    The structural transition in NiAs was studied by neutron diffraction on the single crystalline sample. The crystal structure of NiAs has been reported to be bottom-centered orthorhombic with Cmc2 1 symmetry (niccolite-type). The measurement of temperature dependence of the powder X-ray diffraction revealed that NiAs undergoes a structural transition to the NiAs-type at T t =335 K. In present neutron diffraction experiment at room temperature, we observed the reflections indexed on the basis of the orthorhombic unit cell. The intensities of these reflections are qualitatively explained in terms of the niccolite-type structure with taking account of three domain structures, except for the weak reflections indexed as (001), (003) and (012). Then, the intensities of (001), (002) and (004) reflections were measured in temperature range of 20 to 420 K. The temperature dependences of (002) and (004) reflections qualitatively agree with those of the calculated intensities using the atomic positions of niccolite-type structure. However, the temperature dependence of (001) reflection shows the anomaly around T t , which suggests the symmetry of crystal structure of NiAs is not the Cmc2 1 symmetry. (author)

  19. Impact of recombination on polymorphism of genes encoding Kunitz-type protease inhibitors in the genus Solanum. (United States)

    Speranskaya, Anna S; Krinitsina, Anastasia A; Kudryavtseva, Anna V; Poltronieri, Palmiro; Santino, Angelo; Oparina, Nina Y; Dmitriev, Alexey A; Belenikin, Maxim S; Guseva, Marina A; Shevelev, Alexei B


    The group of Kunitz-type protease inhibitors (KPI) from potato is encoded by a polymorphic family of multiple allelic and non-allelic genes. The previous explanations of the KPI variability were based on the hypothesis of random mutagenesis as a key factor of KPI polymorphism. KPI-A genes from the genomes of Solanum tuberosum cv. Istrinskii and the wild species Solanum palustre were amplified by PCR with subsequent cloning in plasmids. True KPI sequences were derived from comparison of the cloned copies. "Hot spots" of recombination in KPI genes were independently identified by DnaSP 4.0 and TOPALi v2.5 software. The KPI-A sequence from potato cv. Istrinskii was found to be 100% identical to the gene from Solanum nigrum. This fact illustrates a high degree of similarity of KPI genes in the genus Solanum. Pairwise comparison of KPI A and B genes unambiguously showed a non-uniform extent of polymorphism at different nt positions. Moreover, the occurrence of substitutions was not random along the strand. Taken together, these facts contradict the traditional hypothesis of random mutagenesis as a principal source of KPI gene polymorphism. The experimentally found mosaic structure of KPI genes in both plants studied is consistent with the hypothesis suggesting recombination of ancestral genes. The same mechanism was proposed earlier for other resistance-conferring genes in the nightshade family (Solanaceae). Based on the data obtained, we searched for potential motifs of site-specific binding with plant DNA recombinases. During this work, we analyzed the sequencing data reported by the Potato Genome Sequencing Consortium (PGSC), 2011 and found considerable inconsistence of their data concerning the number, location, and orientation of KPI genes of groups A and B. The key role of recombination rather than random point mutagenesis in KPI polymorphism was demonstrated for the first time. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

  20. Production and some properties of crude alkaline proteases of indigenous Central Amazonian rhizobia strains

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    Arlem Nascimento de Oliveira


    Full Text Available Two rhizobia strains isolated from soils of the Central Amazonian floodplain produced appreciable quantities of crude alkaline protease extracts with inexpensive carbon and nitrogen sources. These protease crude extracts were optimally active at pH 9.0-11.0. The optimum temperatures were 35 ºC for Rhizobium sp. strain R-986 and 55 ºC for Bradyrhizobium sp. strain R-993. Protease activities in the crude extracts were enhanced in the presence of 5 mM metal ions, such as Na+, Ca2+, Mg2+ and Mn2+. Rhizobia proteases were strongly inhibited by PMSF, a serine-protease inhibitor. The enzymes were active in the presence of surfactants (SDS and Triton X-100 and stable in oxidizing (H2O2 and reducing agents (β-mercaptoethanol, and organic solvents (acetone, hexane, methanol, 1-propanol and toluene.Duas estirpes de rizóbia isoladas de solos de várzea da Amazônia Central produziram grandes quantidades de proteases alcalinas extracelulares, usando fontes baratas de carbono e nitrogênio. Os extratos brutos de proteases foram ativos em pH 9,0-11,0. As temperaturas ótimas foram de 35 ºC para a enzima do Rhizobium R-986 e de 55 ºC para a do Bradyrhizobium R-993. As atividades proteolíticas aumentaram na presença de 5 mM dos íons Na+, Ca2+ , Mg2+ e Mn2+ . As proteases secretadas pelos rizóbios foram fortemente inibidas por PMSF, um inibidor de serina protease. As enzimas foram ativas na presença de surfactantes (SDS e Triton X-100, e estáveis na presença de agentes oxidantes (H2O2 e redutores (β-mercaptoetanol e solventes orgânicos (acetona, hexano, metanol, 1-propanol e tolueno.

  1. House dust mite major allergens Der p 1 and Der p 5 activate human airway-derived epithelial cells by protease-dependent and protease-independent mechanisms

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    Timmerman J André B


    Full Text Available Abstract House dust mite allergens (HDM cause bronchoconstriction in asthma patients and induce an inflammatory response in the lungs due to the release of cytokines, chemokines and additional mediators. The mechanism how HDM components achieve this is largely unknown. The objective of this study was to assess whether HDM components of Dermatophagoides pteronissinus with protease activity (Der p 1 and unknown enzymatic activity (Der p 2, Der p 5 induce biological responses in a human airway-derived epithelial cell line (A549, and if so, to elucidate the underlying mechanism(s of action. A549 cells were incubated with HDM extract, Der p 1, recombinant Der p 2 and recombinant Der p 5. Cell desquamation was assessed by microscopy. The proinflammatory cytokines, IL-6 and IL-8, were measured by ELISA. Intracellular Ca2+ levels were assessed in A549 cells and in mouse fibroblasts expressing the human protease activated receptor (PAR1, PAR2 or PAR4. HDM extract, Der p 1 and Der p 5 dose-dependently increased the production of IL-6 and IL-8. Added simultaneously, Der p 1 and Der p 5 further increased the production of IL-6 and IL-8. The action of Der p 1 was blocked by cysteine-protease inhibitors, while that of Der p 5 couldn't be blocked by either serine- or cysteine protease inhibitors. Der p 5 only induced cell shrinking, whereas HDM extract and Der p1 also induced cell desquamation. Der p 2 had no effect on A549 cells. Der p 1's protease activity causes desquamation and induced the release of IL6 and IL-8 by a mechanism independent of Ca2+ mobilisation and PAR activation. Der p 5 exerts a protease-independent activation of A549 that involves Ca2+ mobilisation and also leads to the production of these cytokines. Together, our data indicate that allergens present in HDM extracts can trigger protease-dependent and protease-independent signalling pathways in A549 cells.

  2. pH regulation of recombinant glucoamylase production in Fusarium venenatum JeRS 325, a transformant with a Fusarium oxysporum alkaline (trypsin-like) protease promoter. (United States)

    Wiebe, M G; Robson, G D; Shuster, J R; Trinci, A P


    Fusarium venenatum (formerly Fusarium graminearum) JeRS 325 produces heterologous glucoamylase (GAM) under the regulation of a Fusarium oxysporum alkaline (trypsin-like) protease promoter. The glucoamylase gene was used as a reporter gene to study the effects of ammonium and pH on GAM production under the control of the alkaline protease promoter. Between pH 4.0 and 5.8, GAM production in glucose-limited chemostat cultures of JeRS 325 grown at a dilution rate of 0.10 h-1 (doubling time, 6.9 h) on (NH4)2SO4 medium increased in a linear manner with increase in pH. However, at pH 4.0 and below GAM production was almost completely repressed in glucose-limited chemostat cultures grown on (NH4)2SO4 or NaNO3 medium. Thus GAM production in JeRS 325 is regulated by culture pH, not by the nature of the nitrogen source in the medium. The difficulty of using unbuffered medium when investigating putative ammonium repression is also shown. The study demonstrates the potential for use of the alkaline protease promoter in F. graminearum for the production of recombinant proteins in a pH dependent man ner. Copyright 1999 John Wiley & Sons, Inc.

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

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    Porntip Pinlaor

    Full Text Available The liver fluke Opisthorchis viverrini is classified as a class I carcinogen due to the association between cholangiocarcinoma and chronic O. viverrini infection. During its feeding activity within the bile duct, the parasite secretes several cathepsin F cysteine proteases that may induce or contribute to the pathologies associated with hepatobiliary abnormalities.Here, we describe the cDNA, gene organization, phylogenetic relationships, immunolocalization, and functional characterization of the cathepsin F cysteine protease gene, here termed Ov-cf-1, from O. viverrini. The full length mRNA of 1020 nucleotides (nt encoded a 326 amino acid zymogen consisting of a predicted signal peptide (18 amino acids, aa, prosegment (95 aa, and mature protease (213 aa. BLAST analysis using the Ov-CF-1 protein as the query revealed that the protease shared identity with cathepsin F-like cysteine proteases of other trematodes, including Clonorchis sinensis (81%, Paragonimus westermani (58%, Schistosoma mansoni and S. japonicum (52%, and with vertebrate cathepsin F (51%. Transcripts encoding the protease were detected in all developmental stages that parasitize the mammalian host. The Ov-cf-1 gene, of approximately 3 kb in length, included seven exons interrupted by six introns; the exons ranged from 69 to 267 bp in length, the introns from 43 to 1,060 bp. The six intron/exon boundaries of Ov-cf-1 were conserved with intron/exon boundaries in the human cathepsin F gene, although the gene structure of human cathepsin F is more complex. Unlike Ov-CF-1, human cathepsin F zymogen includes a cystatin domain in the prosegment region. Phylogenetic analysis revealed that the fluke, human, and other cathepsin Fs branched together in a clade discrete from the cathepsin L cysteine proteases. A recombinant Ov-CF-1 zymogen that displayed low-level activity was expressed in the yeast Pichia pastoris. Although the recombinant protease did not autocatalytically process and

  4. Protease-sensitive synthetic prions.

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    David W Colby


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

  5. Heterologous expression and characterisation of the Aspergillus aspartic protease involved in the hydrolysis and decolorisation of red-pigmented proteins. (United States)

    Takenaka, Shinji; Umeda, Mayo; Senba, Hisanori; Koyama, Dai; Tanaka, Kosei; Yoshida, Ken-Ichi; Doi, Mikiharu


    Aspergillus repens strain MK82 produces an aspartic protease (PepA_MK82) that efficiently decolorises red-pigmented proteins during dried bonito fermentation. However, further expansion of the industrial applications of PepA_MK82 requires the high-level production and efficient preparation of the recombinant enzyme. The genomic DNA and cDNA fragments encoding the protease were cloned from strain MK82 and sequenced. Phylogenetic analysis of PepA_MK82 and comparisons with previously reported fungal aspartic proteases showed that PepA_MK 82 clusters with different groups of these enzymes. Heterologous expression of PepA_MK82 in Pichia pastoris yielded preparations of higher purity than obtained with an Escherichia coli expression system. Total protease activity in a 100-mL culture of the P. pastoris transformant was 14 times higher than that from an equivalent culture of A. repense MK82. The recombinant PepA_MK82 was easily obtained via acetone precipitation; the final recovery was 83%. PepA_MK82 and its recombinant had similar characteristics in terms of their optimal pH, thermostability, and decolorisation activity. The recombinant was also able to decolorise flaked, dried bonito and to bleach a blood-stained cloth. Given its ability to hydrolyse and decolorise red-pigmented proteins, recombinant PepA_MK8 can be exploited in the food industry and as a stain-removal agent in laundry applications. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  6. Penicillin production in industrial strain Penicillium chrysogenum P2niaD18 is not dependent on the copy number of biosynthesis genes. (United States)

    Ziemons, Sandra; Koutsantas, Katerina; Becker, Kordula; Dahlmann, Tim; Kück, Ulrich


    Multi-copy gene integration into microbial genomes is a conventional tool for obtaining improved gene expression. For Penicillium chrysogenum, the fungal producer of the beta-lactam antibiotic penicillin, many production strains carry multiple copies of the penicillin biosynthesis gene cluster. This discovery led to the generally accepted view that high penicillin titers are the result of multiple copies of penicillin genes. Here we investigated strain P2niaD18, a production line that carries only two copies of the penicillin gene cluster. We performed pulsed-field gel electrophoresis (PFGE), quantitative qRT-PCR, and penicillin bioassays to investigate production, deletion and overexpression strains generated in the P. chrysogenum P2niaD18 background, in order to determine the copy number of the penicillin biosynthesis gene cluster, and study the expression of one penicillin biosynthesis gene, and the penicillin titer. Analysis of production and recombinant strain showed that the enhanced penicillin titer did not depend on the copy number of the penicillin gene cluster. Our assumption was strengthened by results with a penicillin null strain lacking pcbC encoding isopenicillin N synthase. Reintroduction of one or two copies of the cluster into the pcbC deletion strain restored transcriptional high expression of the pcbC gene, but recombinant strains showed no significantly different penicillin titer compared to parental strains. Here we present a molecular genetic analysis of production and recombinant strains in the P2niaD18 background carrying different copy numbers of the penicillin biosynthesis gene cluster. Our analysis shows that the enhanced penicillin titer does not strictly depend on the copy number of the cluster. Based on these overall findings, we hypothesize that instead, complex regulatory mechanisms are prominently implicated in increased penicillin biosynthesis in production strains.

  7. A Naturally Occurring Recombinant Enterovirus Expresses a Torovirus Deubiquitinase. (United States)

    Shang, Pengcheng; Misra, Saurav; Hause, Ben; Fang, Ying


    Enteroviruses (EVs) are implicated in a wide range of diseases in humans and animals. In this study, a novel enterovirus (enterovirus species G [EVG]) (EVG 08/NC_USA/2015) was isolated from a diagnostic sample from a neonatal pig diarrhea case and identified by using metagenomics and complete genome sequencing. The viral genome shares 75.4% nucleotide identity with a prototypic EVG strain (PEV9 UKG/410/73). Remarkably, a 582-nucleotide insertion, flanked by 3C pro cleavage sites at the 5' and 3' ends, was found in the 2C/3A junction region of the viral genome. This insertion encodes a predicted protease with 54 to 68% amino acid identity to torovirus (ToV) papain-like protease (PLP) (ToV-PLP). Structural homology modeling predicts that this protease adopts a fold and a catalytic site characteristic of minimal PLP catalytic domains. This structure is similar to those of core catalytic domains of the foot-and-mouth disease virus leader protease and coronavirus PLPs, which act as deubiquitinating and deISGylating (interferon [IFN]-stimulated gene 15 [ISG15]-removing) enzymes on host cell substrates. Importantly, the recombinant ToV-PLP protein derived from this novel enterovirus also showed strong deubiquitination and deISGylation activities and demonstrated the ability to suppress IFN-β expression. Using reverse genetics, we generated a ToV-PLP knockout recombinant virus. Compared to the wild-type virus, the ToV-PLP knockout mutant virus showed impaired growth and induced higher expression levels of innate immune genes in infected cells. These results suggest that ToV-PLP functions as an innate immune antagonist; enterovirus G may therefore gain fitness through the acquisition of ToV-PLP from a recombination event. IMPORTANCE Enteroviruses comprise a highly diversified group of viruses. Genetic recombination has been considered a driving force for viral evolution; however, recombination between viruses from two different orders is a rare event. In this study, we

  8. Combination treatment with hepatitis C virus protease and NS5A inhibitors is effective against recombinant genotype 1a, 2a, and 3a viruses

    DEFF Research Database (Denmark)

    Gottwein, Judith M; Jensen, Sanne B; Li, Yi-Ping


    With the development of directly acting antivirals, hepatitis C virus (HCV) therapy entered a new era. However, rapid selection of resistance mutations necessitates combination therapy. To study combination therapy in infectious culture systems, we aimed at developing HCV semi-full-length (semi...... to single-drug treatment, combination treatment with relatively low concentrations of asunaprevir and daclatasvir suppressed infection with all five recombinants. Escaped viruses primarily had substitutions at amino acids in the NS3 protease and NS5A domain I reported to be genotype 1 resistance mutations...

  9. The effect of liposome encapsulation on the pharmacokinetics of recombinant secretory leukocyte protease inhibitor (rSLPI) therapy after local delivery to a guinea pig asthma model.

    LENUS (Irish Health Repository)

    Gibbons, Aileen


    Inhaled recombinant Secretory Leukocyte Protease Inhibitor (rSLPI) has shown potential for treatment of inflammatory lung conditions. Rapid inactivation of rSLPI by cathepsin L (Cat L) and rapid clearance from the lungs have limited clinical efficacy. Encapsulation of rSLPI within 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine]:Cholesterol liposomes (DOPS-rSLPI) protects rSLPI against Cat L inactivation in vitro. We aimed to determine the effect of liposomes on rSLPI pharmacokinetics and activity in vitro and after local delivery to the airways in vivo.

  10. Subtype-Specific Differences in Gag-Protease-Driven Replication Capacity Are Consistent with Intersubtype Differences in HIV-1 Disease Progression. (United States)

    Kiguoya, Marion W; Mann, Jaclyn K; Chopera, Denis; Gounder, Kamini; Lee, Guinevere Q; Hunt, Peter W; Martin, Jeffrey N; Ball, T Blake; Kimani, Joshua; Brumme, Zabrina L; Brockman, Mark A; Ndung'u, Thumbi


    There are marked differences in the spread and prevalence of HIV-1 subtypes worldwide, and differences in clinical progression have been reported. However, the biological reasons underlying these differences are unknown. Gag-protease is essential for HIV-1 replication, and Gag-protease-driven replication capacity has previously been correlated with disease progression. We show that Gag-protease replication capacity correlates significantly with that of whole isolates ( r = 0.51; P = 0.04), indicating that Gag-protease is a significant contributor to viral replication capacity. Furthermore, we investigated subtype-specific differences in Gag-protease-driven replication capacity using large well-characterized cohorts in Africa and the Americas. Patient-derived Gag-protease sequences were inserted into an HIV-1 NL4-3 backbone, and the replication capacities of the resulting recombinant viruses were measured in an HIV-1-inducible reporter T cell line by flow cytometry. Recombinant viruses expressing subtype C Gag-proteases exhibited substantially lower replication capacities than those expressing subtype B Gag-proteases ( P identified Gag residues 483 and 484, located within the Alix-binding motif involved in virus budding, as major contributors to subtype-specific replicative differences. In East African cohorts, we observed a hierarchy of Gag-protease-driven replication capacities, i.e., subtypes A/C differences in disease progression. We thus hypothesize that the lower Gag-protease-driven replication capacity of subtypes A and C slows disease progression in individuals infected with these subtypes, which in turn leads to greater opportunity for transmission and thus increased prevalence of these subtypes. IMPORTANCE HIV-1 subtypes are unevenly distributed globally, and there are reported differences in their rates of disease progression and epidemic spread. The biological determinants underlying these differences have not been fully elucidated. Here, we show that

  11. Expression and characterization of recombinant ecarin.

    NARCIS (Netherlands)

    Jonebring, A.; Lange, U.; Bucha, E.; Deinum, J.; Elg, M.; Lovgren, A.


    The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to


    Directory of Open Access Journals (Sweden)

    Eliton Pereira


    Full Text Available This research perform a mapping of teaching spaces of music and dance in the municipalities of Goiânia and Aparecida de Goiânia in the years 2013 and 2014. This project seeks to build a mapping of the professionals schools spaces of music and dance in the metropolitan area of Goiânia, Goiás, Brazil. The research methodology, initially quantitative has been expanded in the pursuit of socio-cultural realities and professional schools of music and dance, where teachers work in this areas.

  13. On-line methanol sensor system development for recombinant ...

    African Journals Online (AJOL)

    On-line methanol sensor system development for recombinant human serum ... of the methanol sensor system was done in a medium environment with yeast cells ... induction at a low temperature and a pH where protease does not function.

  14. Bencana Alam Tak Terhindarkan: Sebuah Tinjauan Pariwisata Nias


    -, Suparwoko


    Indonesian  tourism was grief due to the Bali bombing on October 12, 2002. The tsunami on December 2004 also made the Indonesian tourism was sorrow. The Aceh tsunami devastating some regions of Aceh and North Sumatra had killed more than 125.000 people. The sad feeling from the disaster was not overyet; however, the earthquake in Nias, North Sumatra, was happened just several months after the Aceh tsunami. Because of the Nias victims were more than 500 people, this Nias disaster was proposed ...

  15. Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis. (United States)

    Park, Jong Nam; Park, Sang Kyun; Cho, Min Kyoung; Park, Mi-Kyung; Kang, Shin Ae; Kim, Dong-Hee; Yu, Hak Sun


    In a previous study, we identified an aspartic protease gene (Ts-Asp) from the Trichinella spiralis muscle stage larva cDNA library. The gene sequence of Ts-Asp was 1281 bp long and was found to encode a protein consisting of 405 amino acids, with a molecular mass of 45.248 kD and a pI of 5.95. The deduced Ts-Asp has a conserved catalytic motif with catalytic aspartic acid residues in the active site, a common characteristic of aspartic proteases. In addition, the deduced amino acid sequence of Ts-Asp was found to possess significant homology (above 50%) with aspartic proteases from nematode parasites. Results of phylogenetic analysis indicated a close relationship of Ts-Asp with cathepsin D aspartic proteases. For production of recombinant Ts-Asp (rTs-Asp), the pGEX4T expression system was used. Like other proteases, the purified rTs-Asp was able to digest collagen matrix in vitro. Abundant expression of Ts-Asp was observed in muscle stage larva. Ts-Asp was detected in ES proteins, and was able to elicit the production of specific antibodies. It is the first report of molecular characterization of aspartic protease isolated from T. spiralis. Copyright © 2012 Elsevier B.V. All rights reserved.

  16. Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent


    Roberts, Irma N.; Lam, Xuan Tam; Miranda, Helder; Kieselbach, Thomas; Funk, Christiane


    The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (...

  17. Crystal structure of a novel cysteinless plant Kunitz-type protease inhibitor

    International Nuclear Information System (INIS)

    Hansen, Daiane; Macedo-Ribeiro, Sandra; Verissimo, Paula; Yoo Im, Sonia; Sampaio, Misako Uemura; Oliva, Maria Luiza Vilela


    Bauhinia bauhinioides Cruzipain Inhibitor (BbCI) is a cysteine protease inhibitor highly homologous to plant Kunitz-type inhibitors. However, in contrast to classical Kunitz family inhibitors it lacks cysteine residues and therefore disulfide bridges. BbCI is also distinct in the ability to inactivate enzymes belonging to two different classes, cysteine and serine proteases. Besides inhibiting the cysteine protease cruzipain, BbCI also inhibits cathepsin L and the serine proteases HNE (human neutrophil elastase) and PPE (porcine pancreatic elastase). Monoclinic crystals of the recombinant inhibitor that diffract to 1.7 A resolution were obtained using hanging drop method by vapor diffusion at 18 o C. The refined structure shows the conservative β-trefoil fold features of the Kunitz inhibitors. In BbCI, one of the two characteristic S-S bonds is replaced by the water-mediated interaction between Tyr125 and Gly132. In this work we explore the structural differences between Kunitz-type inhibitors and analyze the essential interactions that maintain the protein structural stability preserving its biological function

  18. Comparative analysis of immune effects in mice model: Clonorchis sinensis cysteine protease generated from recombinant Escherichia coli and Bacillus subtilis spores. (United States)

    Wu, Zhanshuai; Tang, Zeli; Shang, Mei; Zhao, Lu; Zhou, Lina; Kong, Xiangzhan; Lin, Zhipeng; Sun, Hengchang; Chen, Tingjin; Xu, Jin; Li, Xuerong; Huang, Yan; Yu, Xinbing


    Clonorchiasis remains a nonnegligible public health problem in endemic areas. Cysteine protease of Clonorchis sinensis (CsCP) plays indispensable roles in the parasitic physiology and pathology, and has been exploited as a promising drug and vaccine candidate. In recent years, development of spore-based vaccines against multiple pathogens has attracted many investigators' interest. In previous studies, the recombinant Escherichia coli (BL21) and Bacillus subtilis spores expressing CsCP have been successfully constructed, respectively. In this study, the immune effects of CsCP protein purified from recombinant BL21 (rCsCP) and B. subtilis spores presenting CsCP (B.s-CsCP) in Balb/c mice model were conducted with comparative analysis. Levels of specific IgG, IgG1 and IgG2a were significantly increased in sera from both rCsCP and B.s-CsCP intraperitoneally immunized mice. Additionally, recombinant spores expressing abundant fusion CsCP (0.03125 pg/spore) could strongly enhance the immunogenicity of CsCP with significantly higher levels of IgG and isotypes. Compared with rCsCP alone, intraperitoneal administration of mice with spores expressing CsCP achieved a better effect of fighting against C. sinensis infection by slowing down the process of fibrosis. Our results demonstrated that a combination of Th1/Th2 immune responses could be elicited by rCsCP, while spores displaying CsCP prominently induced Th1-biased specific immune responses, and the complex cytokine network maybe mediates protective immune responses against C. sinensis. This work further confirmed that the usage of B. subtilis spores displaying CsCP is an effective way to against C. sinensis.

  19. Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species. (United States)

    Spoerry, Christian; Hessle, Pontus; Lewis, Melanie J; Paton, Lois; Woof, Jenny M; von Pawel-Rammingen, Ulrich


    Recently we have discovered an IgG degrading enzyme of the endemic pig pathogen S. suis designated IgdE that is highly specific for porcine IgG. This protease is the founding member of a novel cysteine protease family assigned C113 in the MEROPS peptidase database. Bioinformatical analyses revealed putative members of the IgdE protease family in eight other Streptococcus species. The genes of the putative IgdE family proteases of S. agalactiae, S. porcinus, S. pseudoporcinus and S. equi subsp. zooepidemicus were cloned for production of recombinant protein into expression vectors. Recombinant proteins of all four IgdE family proteases were proteolytically active against IgG of the respective Streptococcus species hosts, but not against IgG from other tested species or other classes of immunoglobulins, thereby linking the substrate specificity to the known host tropism. The novel IgdE family proteases of S. agalactiae, S. pseudoporcinus and S. equi showed IgG subtype specificity, i.e. IgdE from S. agalactiae and S. pseudoporcinus cleaved human IgG1, while IgdE from S. equi was subtype specific for equine IgG7. Porcine IgG subtype specificities of the IgdE family proteases of S. porcinus and S. pseudoporcinus remain to be determined. Cleavage of porcine IgG by IgdE of S. pseudoporcinus is suggested to be an evolutionary remaining activity reflecting ancestry of the human pathogen to the porcine pathogen S. porcinus. The IgG subtype specificity of bacterial proteases indicates the special importance of these IgG subtypes in counteracting infection or colonization and opportunistic streptococci neutralize such antibodies through expression of IgdE family proteases as putative immune evasion factors. We suggest that IgdE family proteases might be valid vaccine targets against streptococci of both human and veterinary medical concerns and could also be of therapeutic as well as biotechnological use.

  20. Sado, la cerimònia del te japonesa


    Lluch Garcia, Lídia


    La cerimònia del te japonesa, o Sado, és el ritual de la preparació i el servei del te verd japonès, matcha. Influenciada pel budisme zen, és una doctrina que requereix anys d'estudi i pràctica, ja que l'ensenyança d'aquesta cerimònia inclou l'estudi d'altres arts japoneses tals com la cal•ligrafia, o shodo, l'arranjament floral, o ikebana, i l'art del quimono, entre d'altres. Aquest treball explora la història de la cerimònia del te, des dels seus orígens fins a la seva situació actual; així...

  1. The immunological characteristics and probiotic function of recombinant Bacillus subtilis spore expressing Clonorchis sinensis cysteine protease. (United States)

    Tang, Zeli; Shang, Mei; Chen, Tingjin; Ren, Pengli; Sun, Hengchang; Qu, Hongling; Lin, Zhipeng; Zhou, Lina; Yu, Jinyun; Jiang, Hongye; Zhou, Xinyi; Li, Xuerong; Huang, Yan; Xu, Jin; Yu, Xinbing


    Clonorchiasis, a food-borne zoonosis, is caused by Clonorchis sinensis. The intestinal tract and bile ducts are crucial places for C. sinensis metacercariae to develop into adult worms. The endospore of Bacillus subtilis is an ideal oral immunization vehicle for delivery of heterologous antigens to intestine. Cysteine protease of C. sinensis (CsCP) is an endogenous key component in the excystment of metacercariae and other physiological or pathological processes. We constructed a fusion gene of CotC (a coat protein)-CsCP and obtained B. subtilis spores with recombinant plasmid of pEB03-CotC-CsCP (B.s-CotC-CsCP). CotC-CsCP expressed on spores' surface was detected by Western blotting and immunofluorescence. Immunological characteristics of recombinant spore coat protein were evaluated in a mouse model. The levels of CsCP-specific antibodies were detected by ELISA. Effects of recombinant spores on mouse intestine were evaluated by histological staining. The activities of biochemical enzymes in serum were assayed by microplate. Liver sections of infected mice were evaluated by Ishak score after Masson's trichrome. The B.s-CotC-CsCP spores displayed CsCP on their coat. Specific IgG and isotypes were significantly induced by coat proteins of B.s-CotC-CsCP spores after subcutaneous immunization. IgA levels in intestinal mucus and bile of B.s-CotC-CsCP orally treated mice significantly increased. Additionally, more IgA-secreting cells were observed in enteraden and lamina propria regions of the mouse jejunum, and an increased amount of acidic mucins in intestines were also observed. There were no significant differences in enzyme levels of serum among groups. No inflammatory injury was observed in the intestinal tissues of each group. The degree of liver fibrosis was significantly reduced after oral immunization with B.s-CotC-CsCP spores. Bacillus subtilis spores maintained the original excellent immunogenicity of CsCP expressed on their surface. Both local and systemic

  2. Epidemiologia da insônia


    Souza,José Carlos; Reimão,Rubens


    Os autores fizeram uma ampla revisão da literatura científica mundial sobre a epidemiologia da insônia, o mais prevalente dos distúrbios do sono. Foi feito um levantamento bibliográfico, no período de janeiro de 2002 a novembro de 2003, das publicações dos últimos 30 anos, nos bancos de dados MEDLINE e LILACS, sobre a definição e prevalência da insônia, além de suas conseqüências socioeconômicas. As palavras-chaves pesquisadas foram: distúrbio de iniciar o sono, distúrbio de manutenção do son...

  3. Protease Inhibitors Extracted from Caesalpinia echinata Lam. Affect Kinin Release during Lung Inflammation

    Directory of Open Access Journals (Sweden)

    Ilana Cruz-Silva


    Full Text Available Inflammation is an essential process in many pulmonary diseases in which kinins are generated by protease action on kininogen, a phenomenon that is blocked by protease inhibitors. We evaluated kinin release in an in vivo lung inflammation model in rats, in the presence or absence of CeKI (C. echinata kallikrein inhibitor, a plasma kallikrein, cathepsin G, and proteinase-3 inhibitor, and rCeEI (recombinant C. echinata elastase inhibitor, which inhibits these proteases and also neutrophil elastase. Wistar rats were intravenously treated with buffer (negative control or inhibitors and, subsequently, lipopolysaccharide was injected into their lungs. Blood, bronchoalveolar lavage fluid (BALF, and lung tissue were collected. In plasma, kinin release was higher in the LPS-treated animals in comparison to CeKI or rCeEI groups. rCeEI-treated animals presented less kinin than CeKI-treated group. Our data suggest that kinins play a pivotal role in lung inflammation and may be generated by different enzymes; however, neutrophil elastase seems to be the most important in the lung tissue context. These results open perspectives for a better understanding of biological process where neutrophil enzymes participate and indicate these plant inhibitors and their recombinant correlates for therapeutic trials involving pulmonary diseases.

  4. Epidemiologia da insônia Epidemiology of the insomnia

    Directory of Open Access Journals (Sweden)

    José Carlos Souza


    Full Text Available Os autores fizeram uma ampla revisão da literatura científica mundial sobre a epidemiologia da insônia, o mais prevalente dos distúrbios do sono. Foi feito um levantamento bibliográfico, no período de janeiro de 2002 a novembro de 2003, das publicações dos últimos 30 anos, nos bancos de dados MEDLINE e LILACS, sobre a definição e prevalência da insônia, além de suas conseqüências socioeconômicas. As palavras-chaves pesquisadas foram: distúrbio de iniciar o sono, distúrbio de manutenção do sono, distúrbio do despertar precoce, insônia, distúrbios do sono, prevalência da insônia, conseqüências da insônia. Observou-se uma grande variação dos critérios de definição da insônia, dos desenhos metodológicos e dos resultados dos estudos epidemiológicos, o que prejudicou as comparações dos dados das várias pesquisas. As futuras linhas de pesquisa sobre a epidemiologia da insônia devem se embasar em um consenso sobre sua definição e critérios metodológicos mais bem-definidos.A vast literature review on the insomnia epidemiology, the most common sleep disorder, using MEDLINE and LILACS last 30 years of data, was performed from January 2002 to November 2003. The key-words were: sleep initiation disorders, sleep maintenance disorders, early awakening disorder, insomnia, sleep disorders, insomnia prevalence, insomnia consequences. Several insomnia definition criteria and epidemiology researches methods, with data comparison difficulties, were noticed. In the future it will be necessary similar insomnia definition and epidemiology studies criteria.

  5. Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors. (United States)

    Burtoloso, Antonio C B; de Albuquerque, Sérgio; Furber, Mark; Gomes, Juliana C; Gonçalez, Cristiana; Kenny, Peter W; Leitão, Andrei; Montanari, Carlos A; Quilles, José Carlos; Ribeiro, Jean F R; Rocha, Josmar R


    The cysteine protease cruzipain is considered to be a validated target for therapeutic intervention in the treatment of Chagas disease. Anti-trypanosomal activity against the CL Brener strain of T. cruzi was observed in the 0.1 μM to 1 μM range for three nitrile-based cysteine protease inhibitors based on two scaffolds known to be associated with cathepsin K inhibition. The two compounds showing the greatest potency against the trypanosome were characterized by EC50 values (0.12 μM and 0.25 μM) that were an order of magnitude lower than the corresponding Ki values measured against cruzain, a recombinant form of cruzipain, in an enzyme inhibition assay. This implies that the anti-trypanosomal activity of these two compounds may not be explained only by the inhibition of the cruzain enzyme, thereby triggering a putative polypharmacological profile towards cysteine proteases.

  6. Expression, characterization, and cellular localization of knowpains, papain-like cysteine proteases of the Plasmodium knowlesi malaria parasite.

    Directory of Open Access Journals (Sweden)

    Rajesh Prasad

    Full Text Available Papain-like cysteine proteases of malaria parasites degrade haemoglobin in an acidic food vacuole to provide amino acids for intraerythrocytic parasites. These proteases are potential drug targets because their inhibitors block parasite development, and efforts are underway to develop chemotherapeutic inhibitors of these proteases as the treatments for malaria. Plasmodium knowlesi has recently been shown to be an important human pathogen in parts of Asia. We report expression and characterization of three P. knowlesi papain-like proteases, termed knowpains (KP2-4. Recombinant knowpains were produced using a bacterial expression system, and tested for various biochemical properties. Antibodies against recombinant knowpains were generated and used to determine their cellular localization in parasites. Inhibitory effects of the cysteine protease inhibitor E64 were assessed on P. knowlesi culture to validate drug target potential of knowpains. All three knowpains were present in the food vacuole, active in acidic pH, and capable of degrading haemoglobin at the food vacuolar pH (≈5.5, suggesting roles in haemoglobin degradation. The proteases showed absolute (KP2 and KP3 to moderate (KP4 preference for peptide substrates containing leucine at the P2 position; KP4 preferred arginine at the P2 position. While the three knowpains appear to have redundant roles in haemoglobin degradation, KP4 may also have a role in degradation of erythrocyte cytoskeleton during merozoite egress, as it displayed broad substrate specificity and was primarily localized at the parasite periphery. Importantly, E64 blocked erythrocytic development of P. knowlesi, with enlargement of food vacuoles, indicating inhibition of haemoglobin hydrolysis and supporting the potential for inhibition of knowpains as a strategy for the treatment of malaria. Functional expression and characterization of knowpains should enable simultaneous screening of available cysteine protease

  7. Insônia em pacientes com câncer de mama


    Rafihi-Ferreira,Renatha El; Soares,Maria Rita Zoéga


    A insônia é uma queixa frequente em pacientes com câncer de mama, e suas consequências podem causar impacto na qualidade de vida e no curso da doença. O objetivo deste estudo foi revisar a literatura especializada sobre a relação entre insônia e câncer de mama, abordando definição, prevalência, fatores etiológicos e as consequências da insônia no contexto da neoplasia mamária, bem como os tratamentos para insônia nessa população. A busca da literatura ocorreu nos bancos de dados Scopus, Web o...

  8. Generation of an antibody that recognizes Plasmodium chabaudi cysteine protease (chabaupain-1) in both sexual and asexual parasite life cycle and evaluation of chabaupain-1 vaccine potential. (United States)

    Armada, Ana; Gazarini, Marcos L; Gonçalves, Lídia M; Antunes, Sandra; Custódio, Ana; Rodrigues, Armanda; Almeida, António J; Silveira, Henrique; Rosário, Virgílio do; Santos-Gomes, Gabriela; Domingos, Ana


    Malaria cysteine proteases have been shown to be immunogenic and are being exploited as serodiagnostic markers, drug and vaccine targets. Several Plasmodium spp. cysteine proteases have been described and the best characterized of these are the falcipains, a family of papain-family enzymes. Falcipain-2 and falcipain-3 act in concert with other proteases to hydrolyze host erythrocyte hemoglobin in the parasite food vacuole. Falcipain-1 has less similarity to the other falcipains and its physiological role in parasite asexual blood stage still remains uncertain. Immunolocalization studies using an antibody developed against the Plasmodium chabaudi recombinant chabaupain-1, the falcipain-1 ortholog, were performed confirming its cellular localization in both erythrocyte and mosquito ookinete stage. Immunostaining of chabaupain-1 preferentially in apical portion of parasite ookinete suggests that this protease may be related with parasite egression from mosquito midgut. Immune responses to chabaupain-1 were evaluated using two different adjuvants, chitosan nanoparticles and hydroxide aluminum. Mice immunized with the recombinant protein alone or in association with nanoparticles were challenged with P. chabaudi showing that immunization with the recombinant protein confers partial protection to blood stage infection in BALB/c animal model. Copyright © 2013 Elsevier Inc. All rights reserved.

  9. Foot-and-Mouth Disease (FMD) Virus 3C Protease Mutant L127P: Implications for FMD Vaccine Development. (United States)

    Puckette, Michael; Clark, Benjamin A; Smith, Justin D; Turecek, Traci; Martel, Erica; Gabbert, Lindsay; Pisano, Melia; Hurtle, William; Pacheco, Juan M; Barrera, José; Neilan, John G; Rasmussen, Max


    The foot-and-mouth disease virus (FMDV) afflicts livestock in more than 80 countries, limiting food production and global trade. Production of foot-and-mouth disease (FMD) vaccines requires cytosolic expression of the FMDV 3C protease to cleave the P1 polyprotein into mature capsid proteins, but the FMDV 3C protease is toxic to host cells. To identify less-toxic isoforms of the FMDV 3C protease, we screened 3C mutants for increased transgene output in comparison to wild-type 3C using a Gaussia luciferase reporter system. The novel point mutation 3C(L127P) increased yields of recombinant FMDV subunit proteins in mammalian and bacterial cells expressing P1-3C transgenes and retained the ability to process P1 polyproteins from multiple FMDV serotypes. The 3C(L127P) mutant produced crystalline arrays of FMDV-like particles in mammalian and bacterial cells, potentially providing a practical method of rapid, inexpensive FMD vaccine production in bacteria. IMPORTANCE The mutant FMDV 3C protease L127P significantly increased yields of recombinant FMDV subunit antigens and produced virus-like particles in mammalian and bacterial cells. The L127P mutation represents a novel advancement for economical FMD vaccine production. Copyright © 2017 Puckette et al.

  10. Engineered toxins "zymoxins" are activated by the HCV NS3 protease by removal of an inhibitory protein domain.

    Directory of Open Access Journals (Sweden)

    Assaf Shapira

    Full Text Available The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins". In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA and Ricin A chain (RTA, respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.

  11. Effects of anti-tick vaccines, recombinant serine protease inhibitors ...

    African Journals Online (AJOL)

    A preliminary trial of a cocktail of recombinant RAS-1-2 and RIM 36 antigens was conducted in Uganda to assess the effects of ant-tick vaccines against Rhipicephalus appendiculatus tick feeding on Zebu cattle under both experimental and natural conditions. Under experimental conditions, over a period of 28 days, the ...

  12. Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood (United States)

    Yu, Johnson Chung Sing

    Thrombin is a serine protease that plays a key role in the clotting cascade to promote hemostasis following injury to the endothelium. From a clinical diagnostic perspective, in-vivo thrombin activity is linked to various blood clotting disorders, as well as cardiovascular disease (DVT, arteriosclerosis, etc). Thus, the ability to rapidly measure protease activity directly in whole blood will provide important new diagnostics, and clinical researchers with a powerful tool to further elucidate the relationship between circulating protease levels and disease. The ultimate goal is to design novel point of care (POC) diagnostic devices that are capable of monitoring protease activities directly in whole blood and biological sample. A charge-changing substrate specific to the thrombin enzyme was engineered and its functionality was confirmed by a series of experiments. This led to the preliminary design, construction, and testing of two device platforms deemed fully functional for the electrophoretic separation and focusing of charged peptide fragments. The concept of using the existing charge-changing substrate platform for bacterial protease detection was also investigated. Certain strains of E coli are associated with severe symptoms such as abdominal cramps, bloody diarrhea, and vomiting. The OmpT protease is expressed on the outer membrane of E coli and plays a role in the cleavage of antimicrobial peptides, the degradation of recombinant heterologous proteins, and the activation of plasminogen in the host. Thus, a synthetic peptide substrate specific to the OmpT protease was designed and modeled for the purpose of detecting E coli in biological sample.

  13. Serine protease inhibitors containing a Kunitz domain: their role in modulation of host inflammatory responses and parasite survival. (United States)

    de Magalhães, Mariana T Q; Mambelli, Fábio S; Santos, Bruno P O; Morais, Suellen B; Oliveira, Sergio C


    Proteins containing a Kunitz domain have the typical serine protease inhibition function ranging from sea anemone to man. Protease inhibitors play major roles in infection, inflammation disorders and cancer. This review discusses the role of serine proteases containing a Kunitz domain in immunomodulation induced by helminth parasites. Helminth parasites are associated with protection from inflammatory conditions. Therefore, interest has raised whether worm parasites or their products hold potential as drugs for treatment of immunological disorders. Finally, we also propose the use of recombinant SmKI-1 from Schistosoma mansoni as a potential therapeutic molecule to treat inflammatory diseases. Copyright © 2018 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

  14. Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family. (United States)

    Ibrahim, Ahmed Ragaa Nour; Kawamoto, Seiji; Aki, Tsunehiro; Shimada, Yayoi; Rikimaru, Satoshi; Onishi, Nobukazu; Babiker, Elfadil Elfadl; Oiso, Isao; Hashimoto, Kunihiko; Hayashi, Takaharu; Ono, Kazuhisa


    Japanese cedar (Cryptomeria japonica) pollen is a major cause of seasonal pollinosis in Japan. Protease activity in the pollen grains may trigger pro-allergic responses but no such proteases have yet been identified as pollen allergens. We report the molecular cloning and immunochemical characterization of a novel C. japonica pollen allergen belonging to the aspartic protease family. We focused on the C. japonica pollen allergen spot No. 63 (CPA63, 47.5% IgE binding frequency) on our 2-dimensional IgE immunoblot map. The internal amino acid sequences were determined using time-of-flight mass spectrometry. Full-length cpa63 cDNA was cloned by rapid amplification of cDNA ends (RACE)-PCR. Recombinant CPA63 (r-CPA63) was expressed using the baculovirus-insect cell culture system and its IgE binding capacity was analyzed by enzyme-linked immunosorbent assay (ELISA). The proteolytic activity of r-CPA63 was also assessed using a putative mature enzyme produced upon autolysis. cpa63 cDNA encoded a 472 amino acid polypeptide showing about 40% sequence identity to members of the plant atypical aspartic protease family. ELISA showed that r-CPA63 was recognized by IgE antibodies in the serum of 58% (18/31) of Japanese cedar pollinosis patients. We also demonstrated an aspartic protease-like enzyme activity of the putative mature r-CPA63. We have identified the first plant aspartic protease allergen from Japanese cedar pollen. The availability of the CPA63 sequence and its recombinant allergen production system are useful not only for pharmaceutical applications but also for further examination of the role of protease activity in the pathogenesis of cedar pollinosis. 2010 S. Karger AG, Basel.

  15. Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. (United States)

    Yoon, Jaewoo; Maruyama, Jun-ichi; Kitamoto, Katsuhiko


    Proteolytic degradation by secreted proteases into the culture medium is one of the significant problems to be solved in heterologous protein production by filamentous fungi including Aspergillus oryzae. Double (tppA, and pepE) and quintuple (tppA, pepE, nptB, dppIV, and dppV) disruption of protease genes enhanced human lysozyme (HLY) and bovine chymosin (CHY) production by A. oryzae. In this study, we used a quintuple protease gene disruptant and performed successive rounds of disruption for five additional protease genes (alpA, pepA, AopepAa, AopepAd, and cpI), which were previously investigated by DNA microarray analyses for their expression. Gene disruption was performed by pyrG marker recycling with a highly efficient gene-targeting background (∆ligD) as previously reported. As a result, the maximum yields of recombinant CHY and HLY produced by a decuple protease gene disruptant were approximately 30% and 35%, respectively, higher than those produced by a quintuple protease gene disruptant. Thus, we successfully constructed a decuple protease gene disruptant possessing highly improved capability of heterologous protein production. This is the first report on decuple protease gene disruption that improved the levels of heterologous protein production by the filamentous fungus A. oryzae.

  16. Efficient expression and purification of a protease from the common cold virus, human rhinovirus type 14 (United States)

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


    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.

  17. Enhancement in production of recombinant two-chain Insulin Glargine by over-expression of Kex2 protease in Pichia pastoris. (United States)

    Sreenivas, Suma; Krishnaiah, Sateesh M; Govindappa, Nagaraja; Basavaraju, Yogesh; Kanojia, Komal; Mallikarjun, Niveditha; Natarajan, Jayaprakash; Chatterjee, Amarnath; Sastry, Kedarnath N


    Glargine is an analog of Insulin currently being produced by recombinant DNA technology using two different hosts namely Escherichia coli and Pichia pastoris. Production from E. coli involves the steps of extraction of inclusion bodies by cell lysis, refolding, proteolytic cleavage and purification. In P. pastoris, a single-chain precursor with appropriate disulfide bonding is secreted to the medium. Downstream processing currently involves use of trypsin which converts the precursor into two-chain final product. The use of trypsin in the process generates additional impurities due to presence of Lys and Arg residues in the Glargine molecule. In this study, we describe an alternate approach involving over-expression of endogenous Kex2 proprotein convertase, taking advantage of dibasic amino acid sequence (Arg-Arg) at the end of B-chain of Glargine. KEX2 gene over-expression in Pichia was accomplished by using promoters of varying strengths to ensure production of greater levels of fully functional two-chain Glargine product, confirmed by HPLC and mass analysis. In conclusion, this new production process involving Kex2 protease over-expression improves the downstream process efficiency, reduces the levels of impurities generated and decreases the use of raw materials.

  18. Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain (United States)

    Shapira, Assaf; Gal-Tanamy, Meital; Nahary, Limor; Litvak-Greenfeld, Dana; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai


    The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected. PMID:21264238

  19. Enzymatic characteristics of a recombinant neutral protease I (rNpI) from Aspergillus oryzae expressed in Pichia pastoris. (United States)

    Ke, Ye; Huang, Wei-Qian; Li, Jia-zhou; Xie, Ming-quan; Luo, Xiao-chun


    A truncated neutral protease I (NpI) from Aspergillus oryzae 3.042 was expressed in Pichia pastoris with a high enzyme yield of 43101 U/mL. Its optimum pH was about 8.0, and it was stable in the pH range of 5.0-9.0. Its optimum temperature was about 55 °C and retained >90% activity at 50 °C for 120 min. Recombinant NpI (rNpI) was inhibited by Cu(2+) and EDTA. Eight cleavage sites of rNpI in oxidized insulin B-chain were determined by mass spectrometry, and five of them had high hydrophobic amino acid affinity, which makes it efficient in producing antihypertensive peptide IPP from β-casein and a potential debittering agent. The high degree of hydrolysis (DH) of rNpI to soybean protein (8.8%) and peanut protein (11.1%) compared to papain and alcalase makes it a good candidate in the processing of oil industry byproducts. The mutagenesis of H(429), H(433), and E(453) in the deduced zinc-binding motif confirmed rNpI as a gluzincin. All of these results show the great potential of rNpI to be used in the protein hydrolysis industry.

  20. Human eosinophils constitutively express a unique serine protease, PRSS33. (United States)

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


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

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

    International Nuclear Information System (INIS)

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


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

  2. Crystallization and preliminary crystallographic studies of a cysteine protease inhibitor from the human nematode parasite Ascaris lumbricoides

    International Nuclear Information System (INIS)

    Liu, Sanling; Dong, Jianmei; Mei, Guoqiang; Liu, Guiyun; Xu, Wei; Su, Zhong; Liu, Jinsong


    A recombinant cysteine protease inhibitor from the human nematode parasite A. lumbricoides has been overexpressed in E. coli, purified and crystallized. Diffraction data were collected to 2.1 Å resolution. The cysteine protease inhibitor from Ascaris lumbricoides, a roundworm that lives in the human intestine, may be involved in the suppression of human immune responses. Here, the molecular cloning, protein expression and purification, preliminary crystallization and crystallographic characterization of the cysteine protease inhibitor from A. lumbricoides are reported. The rod-shaped crystal belonged to space group C2, with unit-cell parameters a = 99.40, b = 37.52, c = 62.92 Å, β = 118.26°. The crystal diffracted to 2.1 Å resolution and contained two molecules in the asymmetric unit

  3. Mast Cell Proteases 6 and 7 Stimulate Angiogenesis by Inducing Endothelial Cells to Release Angiogenic Factors.

    Directory of Open Access Journals (Sweden)

    Devandir Antonio de Souza Junior

    Full Text Available Mast cell proteases are thought to be involved with tumor progression and neo-vascularization. However, their exact role is still unclear. The present study was undertaken to further elucidate the function of specific subtypes of recombinant mouse mast cell proteases (rmMCP-6 and 7 in neo-vascularization. SVEC4-10 cells were cultured on Geltrex® with either rmMCP-6 or 7 and tube formation was analyzed by fluorescence microscopy and scanning electron microscopy. Additionally, the capacity of these proteases to induce the release of angiogenic factors and pro and anti-angiogenic proteins was analyzed. Both rmMCP-6 and 7 were able to stimulate tube formation. Scanning electron microscopy showed that incubation with the proteases induced SVEC4-10 cells to invade the gel matrix. However, the expression and activity of metalloproteases were not altered by incubation with the mast cell proteases. Furthermore, rmMCP-6 and rmMCP-7 were able to induce the differential release of angiogenic factors from the SVEC4-10 cells. rmMCP-7 was more efficient in stimulating tube formation and release of angiogenic factors than rmMCP-6. These results suggest that the subtypes of proteases released by mast cells may influence endothelial cells during in vivo neo-vascularization.

  4. Insónia e hipertensão


    Santos, Pedro Duarte Lourenço dos


    Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz A insónia e hipertensão são duas patologias prevalentes na sociedade atual, sendo que ambas são problemas crescentes no âmbito da saúde pública. O efeito isolado de ambas as doenças tem interesse social, laboral e económico. No entanto o interesse científico destas patologias é acrescido com o fenómeno de causalidade entre insónia e hipertensão. Apesar de serem patologias distintas entre si, a...

  5. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage. (United States)

    Pettit, Steven C; Gulnik, Sergei; Everitt, Lori; Kaplan, Andrew H


    Activation of the human immunodeficiency virus type 1 (HIV-1) protease is an essential step in viral replication. As is the case for all retroviral proteases, enzyme activation requires the formation of protease homodimers. However, little is known about the mechanisms by which retroviral proteases become active within their precursors. Using an in vitro expression system, we have examined the determinants of activation efficiency and the order of cleavage site processing for the protease of HIV-1 within the full-length GagPol precursor. Following activation, initial cleavage occurs between the viral p2 and nucleocapsid proteins. This is followed by cleavage of a novel site located in the transframe domain. Mutational analysis of the dimer interface of the protease produced differential effects on activation and specificity. A subset of mutations produced enhanced cleavage at the amino terminus of the protease, suggesting that, in the wild-type precursor, cleavages that liberate the protease are a relatively late event. Replacement of the proline residue at position 1 of the protease dimer interface resulted in altered cleavage of distal sites and suggests that this residue functions as a cis-directed specificity determinant. In summary, our studies indicate that interactions within the protease dimer interface help determine the order of precursor cleavage and contribute to the formation of extended-protease intermediates. Assembly domains within GagPol outside the protease domain also influence enzyme activation.

  6. Earthworm Protease

    Directory of Open Access Journals (Sweden)

    Rong Pan


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

  7. Earthworm Protease

    International Nuclear Information System (INIS)

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


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

  8. Genomic and exoproteomic analyses of cold- and alkaline-adapted bacteria reveal an abundance of secreted subtilisin-like proteases. (United States)

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


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

  9. Optimizing HIV-1 protease production in Escherichia coli as fusion protein

    Directory of Open Access Journals (Sweden)

    Piubelli Luciano


    Full Text Available Abstract Background Human immunodeficiency virus (HIV is the etiological agent in AIDS and related diseases. The aspartyl protease encoded by the 5' portion of the pol gene is responsible for proteolytic processing of the gag-pol polyprotein precursor to yield the mature capsid protein and the reverse transcriptase and integrase enzymes. The HIV protease (HIV-1Pr is considered an attractive target for designing inhibitors which could be used to tackle AIDS and therefore it is still the object of a number of investigations. Results A recombinant human immunodeficiency virus type 1 protease (HIV-1Pr was overexpressed in Escherichia coli cells as a fusion protein with bacterial periplasmic protein dithiol oxidase (DsbA or glutathione S-transferase (GST, also containing a six-histidine tag sequence. Protein expression was optimized by designing a suitable HIV-1Pr cDNA (for E. coli expression and to avoid autoproteolysis and by screening six different E. coli strains and five growth media. The best expression yields were achieved in E. coli BL21-Codon Plus(DE3-RIL host and in TB or M9 medium to which 1% (w/v glucose was added to minimize basal expression. Among the different parameters assayed, the presence of a buffer system (based on phosphate salts and a growth temperature of 37°C after adding IPTG played the main role in enhancing protease expression (up to 10 mg of chimeric DsbA:HIV-1Pr/L fermentation broth. GST:HIVPr was in part (50% produced as soluble protein while the overexpressed DsbA:HIV-1Pr chimeric protein largely accumulated in inclusion bodies as unprocessed fusion protein. A simple refolding procedure was developed on HiTrap Chelating column that yielded a refolded DsbA:HIV-1Pr with a > 80% recovery. Finally, enterokinase digestion of resolubilized DsbA:HIV-1Pr gave more than 2 mg of HIV-1Pr per liter of fermentation broth with a purity ≤ 80%, while PreScission protease cleavage of soluble GST:HIVPr yielded ~ 0.15 mg of pure HIV-1

  10. Millora en el tractament contra la miastènia gravis


    Ponseti, J.M.


    La miastènia gravis és una malaltia autoimmune i crónica, caracteritzada per una gran debilitat muscular. Un assaig clínic realitzat per la Unitat de Miastènia de l'Hospital General Universitari Vall d'Hebron durant cinc anys acaba de demostrar els beneficis d'incloure un fàrmac, el tacrolimus, per guarir aquesta malaltia.

  11. A produção do espaço intra-urbano de aparecida de Goiânia e a dinâmica metropolitana de Goiânia: de 1960 aos anos 2000


    SANTOS, Lucas Maia dos


    Aparecida de Goiânia é uma cidade que se desenvolve numa relação inextricável com Goiânia. O objetivo desta pesquisa foi justamente compreender a papel desta relação no processo de estruturação do espaço intra-urbano aparecidense. A questão central que norteou nossas análises foi: como se formou Aparecida de Goiânia? A esta pergunta seminal, somam-se outras: a) quais os processos, os atores e as etapas do processo de produção do espaço intra-urbano de Aparecida de Goiânia? c) quais as implica...

  12. Amazônia: a vision that emerges from the waters

    Directory of Open Access Journals (Sweden)

    Ricardo José Nogueira


    Full Text Available O objetivo do texto é fazer emergir do pesado manto do discurso acadêmico uma outra visáo da Amazonia, não a dos holofotes dos grandes eventos que incandescem o observador, impedindo-o de ver além de sua luz, mas de urna Amazonia de acontecimentos quase imperceptíveis que lhe dão vida. Uma Amazônia que acontece nos vales fluviais, a Amazônia das várzeas, e ainda preserva um modo de vida peculiar, extremamente dinâmico e com rede própria de relações. Uma Amazônia que permanece na sombra. Ou na água, esquecida ou negada com a máscara do atraso para ser superada. Que contudo teima em existir, e não se constitui, para o discurso hegemônico, como foco ou vítima da ação dos atores dominantes. É dessa Amazônia que o texto fala, dando-lhe uma visáo de dentro, do seu âmago. Uma visão que emerge das águas...

  13. New tick records in Rondônia, Western Brazilian Amazon. (United States)

    Labruna, Marcelo Bahia; Barbieri, Fábio Silva; Martins, Thiago Fernandes; Brito, Luciana Gatto; Ribeiro, Francisco Dimas Sales


    In the present study, we provide new tick records from Vilhena Municipality, in the Southeast of the State of Rondônia, Northern Brazil. Ticks collected from a capybara, Hydrochoerus hydrochaeris (Linnaeus), were identified as Amblyomma romitii Tonelli-Rondelli (1 female), and Amblyomma sp. (1 larva). Ticks collected from a harpy eagle, Harpia harpyja (Linnaeus), were identified as Amblyomma cajennense (Fabricius) (16 nymphs) and Haemaphysalis juxtakochi Cooley (1 nymph). Ticks collected from a yellow-footed tortoise, Chelonoidis denticulada (Linnaeus), were identified as Amblyomma rotundatum Koch (10 females, 2 nymphs), and Amblyomma sp. (2 larvae). The present record of A. romitii is the first in the State of Rondônia, and represents the southernmost record for this tick species, indicating that its distribution area is much larger than currently recognized. Although both A. cajennense and H. juxtakochi have been reported parasitizing various bird species, we provide the first tick records on a harpy eagle. A. rotundatum is widespread in the State of Rondônia, and has been previously reported on the yellow-footed tortoise. The present records increase the tick fauna of Rondônia to 26 species.

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

    DEFF Research Database (Denmark)

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


    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...

  15. Co-evolution of insect proteases and plant protease inhibitors. (United States)

    Jongsma, Maarten A; Beekwilder, Jules


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

  16. Amino acid sequence requirements in the human IgA1 hinge for cleavage by streptococcal IgA1 proteases

    DEFF Research Database (Denmark)

    Senior, BW; Batten, MR; Kilian, Mogens


    All the IgA1 proteases of the different pathogenic species of Streptococcus cleave the hinge of the alpha chain of human IgA1 only at one proline-threonine peptide bond. In order to study the importance of these amino acids for cleavage, several hinge mutant recombinant IgA1 antibodies were const...... constructed. The mutations were found to be without major effect upon the structure or functional abilities of the antibodies. However, they had a major effect upon their sensitivity to cleavage by some of the IgA1 proteases....

  17. Aragón, Luis E. (2013), Amazônia: conhecer para desenvolver e conservar. Cinco temas para um debate


    Corbin, Hisakhana Pahoona


    Neste livro discutem-se cinco questões, cada uma num capítulo, com o propósito de incitar debates sobre os grandes desafios ambientais, sociais e econômicos que envolvem o desenvolvimento da Amazônia: 1. Amazônia, conceito polissêmico?; 2. Amazônia vazio demográfico?; 3. Amazônia, terra incógnita?; 4. Migração internacional, nova descoberta da Amazônia?; 5. Há futuro para o desenvolvimento sustentável na Amazônia? Esses questionamentos, argumenta o autor, “alicerçam-se na premissa de que o co...

  18. Recombinant immobilized rhizopuspepsin as a new tool for protein digestion in hydrogen/deuterium exchange mass spectrometry

    Czech Academy of Sciences Publication Activity Database

    Rey, M.; Man, Petr; Brandolin, G.; Forest, E.; Pelosi, L.


    Roč. 23, č. 21 (2009), s. 3431-3438 ISSN 0951-4198 Institutional research plan: CEZ:AV0Z50200510 Keywords : mass spectrometry * recombinant immobilized rhizopuspepsin * protease Subject RIV: CE - Biochemistry Impact factor: 2.695, year: 2009

  19. A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity. (United States)

    Nautiyal, Kalpana; Kuroda, Yutaka


    Tobacco Etch Virus (TEV) protease is used in the purification of recombinant proteins, but its usage is often hampered by solubility issues. Here, we report a short, 12-residue solubility enhancing peptide (SEP) tag attached at the C-terminus of TEV (TEV-C9R). We assessed the effects of the C9R tag on the biophysical and biochemical characteristics of TEV. The yield of HPLC purified TEV-C9R expressed in E. coli grown in 200 mL LB or TB media was between 10 and 13 mg, which was up to 6.5 times higher than the yield of the untagged TEV (untagged-TEV). TEV-C9R was active over a pH range of 5-8, which was wider than that of the commonly used thrombin, and it remained active upon incubation at 60 °C much longer than the untagged-TEV, which aggregated at this temperature. Static and dynamic light scattering demonstrated the higher solubility of purified TEV-C9R. Furthermore, the thermal unfolding of TEV-C9R, as assessed by circular dichroism at pH 4.7, was almost perfectly reversible, in contrast to that of untagged-TEV, which aggregated at high temperature. These results demonstrate the improved biophysical and biochemical characteristics of TEV-C9R originating from higher solubility and provide another example of how SEP tags can enhance enzyme solubility without altering its activity. Copyright © 2018 Elsevier B.V. All rights reserved.

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

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


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

  1. The mimivirus R355 gene product: preliminary crystallographic analysis of a putative ubiquitin-like protein-specific protease

    International Nuclear Information System (INIS)

    Jeudy, Sandra; Lartigue, Audrey; Mansuelle, Pascal; Ogata, Yuki; Abergel, Chantal


    The genome sequence of mimivirus, the largest known double-stranded DNA virus, encodes a putative protease: the R355 gene product. Its expression in E. coli, its crystallization and the preliminary phasing of a MAD data set using the selenium signal present in a crystal of recombinant selenomethionine-substituted protein are reported. The complete genome sequence of the largest known double-stranded DNA virus, mimivirus, reveals the presence of a gene (denoted R355) that potentially encodes a cysteine protease that is expressed late (after 6 h) in the infectious cycle of the virus. In order to verify a sequence-based functional prediction and understand its role during the infectious process, the R355 protein was produced to assay its proteolytic activity and solve its three-dimensional structure. Here, the preliminary crystallographic analysis of the recombinant viral protein is reported. The crystals belonged to the orthorhombic space group P2 1 2 1 2 1 , with a monomer in the asymmetric unit. A MAD data set was used for preliminary phasing using the selenium signal from a selenomethionine-substituted protein crystal

  2. Amb M. Antònia Canals al Nepal de Viki Sherpa


    Siqués, Carina


    Relació del viatge fet per la Maria Antònia Canals a Nepal amb motiu de l’acte de celebració de la signatura del conveni de col·laboració entre la Universitat de Tribhuvan a Katmandu i la Universitat de Vic per crear el centre de formació del professorat d’infantil i de primària M. Antònia Canals

  3. Amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by streptococcal IgA1 proteases

    DEFF Research Database (Denmark)

    Batten, MR; Senior, BW; Kilian, Mogens


    The amino acid sequence requirements in the hinge of human immunoglobulin A1 (IgA1) for cleavage by IgA1 proteases of different species of Streptococcus were investigated. Recombinant IgA1 antibodies were generated with point mutations at proline 227 and threonine 228, the residues lying on either...... side of the peptide bond at which all streptococcal IgA1 proteases cleave wild-type human IgA1. The amino acid substitutions produced no major effect upon the structure of the mutant IgA1 antibodies or their functional ability to bind to Fcalpha receptors. However, the substitutions had a substantial...... effect upon sensitivity to cleavage with some streptococcal IgA1 proteases, with, in some cases, a single point mutation rendering the antibody resistant to a particular IgA1 protease. This effect was least marked with the IgA1 protease from Streptococcus pneumoniae, which showed no absolute requirement...

  4. Novos relatos de carrapatos em Rondônia, Amazônia ocidental brasileira


    LABRUNA, Marcelo Bahia; BARBIERI, Fábio Silva; MARTINS, Thiago Fernandes; BRITO, Luciana Gatto; RIBEIRO, Francisco Dimas Sales


    In the present study, we provide new tick records from Vilhena Municipality, in the Southeast of the State of Rondônia, Northern Brazil. Ticks collected from a capybara, Hydrochoerus hydrochaeris (Linnaeus), were identified as Amblyomma romitii Tonelli-Rondelli (1 female), and Amblyomma sp. (1 larva). Ticks collected from a harpy eagle, Harpia harpyja (Linnaeus), were identified as Amblyomma cajennense (Fabricius) (16 nymphs) and Haemaphysalis juxtakochi Cooley (1 nymph). Ticks collected from...

  5. Insônia primária: diagnóstico diferencial e tratamento


    Monti,Jaime M


    A insônia primária é uma dissonia caracterizada pela dificuldade em iniciar e/ou manter o sono e pela sensação de não ter um sono reparador durante um período não inferior a 1 mês. Do ponto de vista polissonográfico, é acompanhada de alterações na indução, na continuidade e na estrutura do sono. Geralmente aparece no adulto jovem, é mais freqüente na mulher e tem um desenvolvimento crônico. A insônia primária é observada de 12,5% a 22,2% dos pacientes portadores de insônia crônica, sendo prec...

  6. Principais políticas de fomento do turismo na Amazônia: análise dos primeiros planos de turismo da Amazônia (PTA I e II e do PROECOTUR

    Directory of Open Access Journals (Sweden)

    Kassia Suelen da Silva Farias


    Full Text Available Verifica-se que o turismo tem passado por inúmeras transformações no que concerne ao planejamento e gestão da atividade. Observa-se que desde a década de 1970 tem-se pensado em diferentes modelos de desenvolvimento de turismo para a Amazônia, almejando o progresso econômico e social. Neste processo, na década de 1970, a Superintendência de Desenvolvimento da Amazônia (SUDAM, deu inicio a discursão de criação do primeiro Plano de Turismo da Amazônia (PTA que deu inicio na década de 1980 até 1985. A partir da década de 1990 as estratégias de turismo para a região foram desenvolvidas em conformidade com a preocupação mundial em torno da natureza e do homem. Produto deste contexto, o II PTA foi lançado com o objetivo principal de desenvolver medidas que conciliassem o desenvolvimento econômico às questões ambientais e sociais. Outra ação também importante para esse período foi a promoção do desenvolvimento regional por meio de polos de turismo integrados, conforme determinava o PROECOTUR. O objetivo deste artigo, portanto, consiste em destacar as primeiras políticas públicas de turismo para o desenvolvimento dos estados e municípios da Amazônia que deram inicio a partir de 1970. Para isso, a estruturação conceitual do trabalho fundamentou-se em políticas públicas e planejamento de turismo na Amazônia. Utiliza-se como metodologia a pesquisa documental, por meio de diagnóstico que enfatiza os principais resultados obtidos pelos I e II Planos de Turismo da Amazônia, a partir da interpretação e análise documental; O artigo também adota a pesquisa bibliográfica, pois analisa as principais estratégias de desenvolvimento do turismo na Amazônia. A partir de tal análise, conclui-se que as mudanças políticas de turismo ocorridas na Amazônia entre a década de 1970 e 1990 - apesar de estarem acompanhadas por ações de planejamento do governo nacional e até mesmo pelas tendências mundiais - n

  7. Aspartic Protease Zymography Case Study: Detection of Fungal Acid Proteases by Zymography. (United States)

    Kernaghan, Gavin; Mayerhofer, Michael


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

  8. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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


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

  9. Genome Sequence of Lactobacillus salivarius NIAS840, Isolated from Chicken Intestine (United States)

    Ham, Jun-Sang; Kim, Hyoun-Wook; Seol, Kuk-Hwan; Jang, Aera; Jeong, Seok-Geun; Oh, Mi-Hwa; Kim, Dong-Hun; Kang, Dae-Kyung; Kim, Geun-Bae; Cha, Chang-Jun


    Lactobacillus salivarius is a well-known lactic acid bacterium to which increasing attention has been paid recently for use as probiotics for humans and animals. L. salivarius NIAS840 was first isolated from broiler chicken feces, displaying antimicrobial activities against multidrug-resistant Staphylococcus aureus and Salmonella enterica serovar Typhimurium. Here, we report the genome sequence of L. salivarius NIAS840 (2,046,557 bp) including a small plasmid and two megaplasmids. PMID:21914873

  10. Proteases and protease inhibitors of urinary extracellular vesicles in diabetic nephropathy. (United States)

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


    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.

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

    Directory of Open Access Journals (Sweden)

    Luca Musante


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

  12. Epidemiologia da insônia Epidemiology of the insomnia


    José Carlos Souza; Rubens Reimão


    Os autores fizeram uma ampla revisão da literatura científica mundial sobre a epidemiologia da insônia, o mais prevalente dos distúrbios do sono. Foi feito um levantamento bibliográfico, no período de janeiro de 2002 a novembro de 2003, das publicações dos últimos 30 anos, nos bancos de dados MEDLINE e LILACS, sobre a definição e prevalência da insônia, além de suas conseqüências socioeconômicas. As palavras-chaves pesquisadas foram: distúrbio de iniciar o sono, distúrbio de manutenção do son...

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

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

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

    International Nuclear Information System (INIS)

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


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

  15. The crystal structure of recombinant proDer p 1, a major house dust mite proteolytic allergen

    DEFF Research Database (Denmark)

    Meno, Kåre; Thorsted, Peter B; Ipsen, Henrik


    Allergy to house dust mite is among the most prevalent allergic diseases worldwide. Most house dust mite allergic patients react to Der p 1 from Dermatophagoides pteronyssinus, which is a cysteine protease. To avoid heterogeneity in the sample used for crystallization, a modified recombinant...

  16. Diferença no tamanho de cascas de ovos de colônias de Panstrongylus megistus

    Directory of Open Access Journals (Sweden)

    Rosa João A da


    Full Text Available O objetivo do estudo foi avaliar as mensurações de cascas de ovos de duas colônias laboratoriais e uma silvestre de P. megistus. De cada uma das colônias, 50 cascas de ovos foram mensuradas por meio de microscópio estereoscópico e sistema de análise de imagem QWin. A análise estatística foi efetuada utilizando o teste t-Student, não pareado no programa INSTAT. A largura, o comprimento e o diâmetro da abertura opercular das cascas de ovos de uma colônia silvestre foram maiores que de duas colônias laboratoriais de P. megistus.

  17. Bicistronic expression plasmid for the rapid production of recombinant fused proteins in Escherichia coli. (United States)

    Yero, Daniel; Pajón, Rolando; Niebla, Olivia; Sardiñas, Gretel; Vivar, Isbel; Perera, Yasser; García, Darien; Delgado, Maité; Cobas, Karem


    In the post-genomic era, every aspect of the production of proteins must be accelerated. In this way, several vectors are currently exploited for rapid production of recombinant proteins in Escherichia coli. N-terminal fusions to the first 47 amino acids of the LpdA (dihydrolipoamide dehydrogenase A) protein of Neisseria meningitidis have been shown to increase the expression of recombinant proteins. Consequently, we have constructed a modified N-terminal LpdA fusion vector, introducing the blue/white colony selection by exploiting a bicistronic gene organization. In the new vector, the sequence encoding the first 47 amino acids of meningococcal LpdA and the alpha-peptide sequence of beta-galactosidase were connected via a ribosome-binding site, and two MCSs (multiple cloning sites) were located surrounding the latter, allowing efficient cloning by colour selection of recombinants. The vector was also improved with the addition of a C-terminal polyhistidine tag, and an EKS (enterokinase recognition sequence) immediately after the LpdA fusion sequence. The new plasmid was employed in the expression and purification of six different bacterial polypeptides. One of these recombinant proteins, P6 protein from Haemophilus influenzae, was used as a model and its N-terminal fusion sequence was totally removed from the recombinant version after incubation with the enterokinase protease, while the polyhistidine tail successfully allowed the purification of the unfused protein from the protease reaction. Two completely new neisserial vaccine candidates, NMB0088 and NMB1126 proteins, were cloned, expressed and purified using this system. To our knowledge, this constitutes the first report of the cloning and expression of these proteins in E. coli.

  18. High level of expression of recombinant human granulocyte-macrophage colony stimulating factor in transgenic rice cell suspension culture

    DEFF Research Database (Denmark)

    Shin, Yun-Ji; Hong, Shin-Young; Kwon, Tae-Ho


    Recombinant human granulocyte-macrophage colony stimulating factor (hGM-CSF) has been previously produced in tobacco cell suspension cultures. However, the amount of hGM-CSF accumulated in the culture medium dropped quickly from its maximum of 150 microg/L at 5 d after incubation. To overcome...... of recombinant hGM-CSF in transgenic rice cell suspension culture and protease activity of this culture medium was low compared to that of tobacco culture system....

  19. Activity, specificity, and probe design for the smallpox virus protease K7L. (United States)

    Aleshin, Alexander E; Drag, Marcin; Gombosuren, Naran; Wei, Ge; Mikolajczyk, Jowita; Satterthwait, Arnold C; Strongin, Alex Y; Liddington, Robert C; Salvesen, Guy S


    The K7L gene product of the smallpox virus is a protease implicated in the maturation of viral proteins. K7L belongs to protease Clan CE, which includes distantly related cysteine proteases from eukaryotes, pathogenic bacteria, and viruses. Here, we describe its recombinant high level expression, biochemical mechanism, substrate preference, and regulation. Earlier studies inferred that the orthologous I7L vaccinia protease cleaves at an AG-X motif in six viral proteins. Our data for K7L suggest that the AG-X motif is necessary but not sufficient for optimal cleavage activity. Thus, K7L requires peptides extended into the P7 and P8 positions for efficient substrate cleavage. Catalytic activity of K7L is substantially enhanced by homodimerization, by the substrate protein P25K as well as by glycerol. RNA and DNA also enhance cleavage of the P25K protein but not of synthetic peptides, suggesting that nucleic acids augment the interaction of K7L with its protein substrate. Library-based peptide preference analyses enabled us to design an activity-based probe that covalently and selectively labels K7L in lysates of transfected and infected cells. Our study thus provides proof-of-concept for the design of inhibitors and probes that may contribute both to a better understanding of the role of K7L in the virus life cycle and the design of novel anti-virals.

  20. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne


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

  1. Pengaruh Budaya Patriarki Terhadap Partisipasi Politik Perempuan Di Dprd Kabupaten Nias Pada Pemilihan Legislatif Tahun 2014


    Halawa, Nota Patrit Karsa


    This research review the facts about the influence of patriarchal culture to women's participation in Nias regency on the legislative elections in 2014. The background of this research because seeinga very strong patriarchal culture in Nias district looks of social and cultural life of society as the system of patrilineal, traditional feast and the inheritance that prefers men, This is a great impact on the development of women's quality of Nias, especially in politics. The theory used i...

  2. Sedative antidepressants and insomnia Antidepressivos sedativos e insônia

    Directory of Open Access Journals (Sweden)

    Walter André dos Santos Moraes


    Full Text Available OBJECTIVE: The present review addresses the relationship between sleep and depression and how serotonergic transmission is implicated in both conditions. METHOD: Literature searches were performed in the PubMed and MedLine databases up to March 2010. The terms searched were "insomnia", "depression", "sedative antidepressants" and "serotonin". In order to pinpoint the sedative antidepressants most used to treat insomnia, 34 ISI articles, mainly reviews and placebo-controlled clinical trials, were selected from 317 articles found in our primary search. RESULTS: Sleep problems may appear months before the diagnosis of clinical depression and persist after the resolution of depression. Treatment of insomnia symptoms may improve this comorbid disease. Some antidepressant drugs can also result in insomnia or daytime sleepiness. Serotonin (5-HT demonstrates a complex pattern with respect to sleep and wakefulness that is related to the array of 5-HT receptor subtypes involved in different physiological functions. It is now believed that 5HT2 receptor stimulation is subjacent to insomnia and changes in sleep organization related to the use of some antidepressants. CONCLUSION: Some drugs commonly prescribed for the treatment of depression may worsen insomnia and impair full recovery from depression. 5-HT2 receptor antagonists are promising drugs for treatment strategies since they can improve comorbid insomnia and depression.OBJETIVO: Esta atualização aborda a relação entre sono e depressão e como a transmissão serotoninérgica está envolvida em ambas condições. MÉTODO: Foi realizada uma busca na literatura no PubMed e MedLine até março de 2010 com os termos "insônia", "depressão", "antidepressivos sedativos" e "serotonina". A fim de contemplar os antidepressivos sedativos mais utilizados no tratamento da insônia, 34 artigos ISI, principalmente revisões e estudos clínicos placebo-controlados, foram selecionados entre 317 artigos

  3. Epidemiologic characterization of bovine tuberculosis in the State of Rondônia, Brazil

    Directory of Open Access Journals (Sweden)

    Fabiano Benitez Vendrame


    Full Text Available A cross sectional study was performed between June 2009 and March 2010 to determine the situation of bovine tuberculosis (bTB in Rondônia. The state was divided into three regions and, in each of them, 300 farms with reproductive activity were randomly chosen and considered as primary sample units. In the selected farms, an epidemiologic questionnaire was applied. A fixed number of bovine females older than two years of age was randomly selected and tested through comparative cervical tuberculin test. Considering the State of Rondônia, the apparent prevalence of bTB positive farms was 2.3% (95% CI = 1.5–3.5%. The prevalence in the regions varied from 1.7% (95% CI = 0.7 – 4% to 3% (95% CI = 1.6–5.7%. The apparent prevalence of bTB positive animals in the State of Rondônia was 0.12% (95% CI = 0.06–0.25% and varied from 0.08% (95% CI = 0.04–0.18% to 0.15% (95% CI = 0.07–0.33% in the regions. The risk factor associated to tuberculosis in the State of Rondônia was the acquisition of animals (OR = 7.1; 95% CI = 1.6–31.1. The State of Rondônia should implement a surveillance system to detect bTB-infected herds to certify them as bTB-free. Moreover, an efficient health education program to inform farmers to test replacement animals for bTB prior to introduction in their herds should also be implemented.

  4. Functional, Responsive Materials Assembled from Recombinant Oleosin (United States)

    Hammer, Daniel

    Biological cells are surrounded by a plasma membrane made primarily of phospholipids that form a bilayer. This membrane is permselective and compartmentalizes the cell. A simple form of artificial cell is the vesicle, in which a phospholipid bilayer membrane surrounds an aqueous solution. However, there is no a priori reason why a membrane needs to be made of phospholipids. It could be made of any surfactant that forms a bilayer. We have assembled membranes and other structures from the recombinant plant protein oleosin. The ability to assemble from a recombinant protein means that every molecule is identical, we have complete control over the sequence, and hence can build in designer functionality with high fidelity, including adhesion and enzymatic activity. Such incorporation is trivial using the tools of molecular biology. We find that while many variants of oleosin make membranes, others make micelles and sheets. We show how the type of supramolecular structure can be altered by the conditions of solvent, such as ionic strength, and the architecture of the surfactant itself. We show that protease cleavable domains can be incorporated within oleosin, and be engineered to protect other functional domains such as adhesive motifs, to make responsive materials whose activity and shape depend on the action of proteases. We will also present the idea of making ``Franken''-oleosins, where large domains of native oleosin are replaced with domains from other functional proteins, to make hybrids conferred by the donor protein. Thus, we can view oleosin as a template upon which a vast array of designer functionalities can be imparted..

  5. Mapping interpersonal violence against women in the District of Nias, 2009-2016

    Directory of Open Access Journals (Sweden)

    Mita Yesyca


    each sub-district and is highly dependent on regional geographic-economic conditions. Therefore, the approach to the resolution of violence in each sub-district may vary. As a conclusion, the practice of violence against women in the Nias District is like an iceberg phenomenon that still needs to be followed up. The research also shows the potential of related institutions partnership, especially encouraged by churches, to end practices of interpersonal violence against women in the Nias District.

  6. Gender inequality : Behind maternal mortality in Nias Island, North Sumatra, Indonesia: Towards a gender audit

    NARCIS (Netherlands)

    Zaluchu, F.


    Singling out Nias Island in North Sumatra Indonesia as the research location, this research attempts to analyze local aspects pertaining to gender inequality in the context of maternal health in Indonesia. This research aims to portray the presence of gender inequality in Nias, and how it affects

  7. Cloning, characterization, expression and antifungal activity of an alkaline serine protease of Aureobasidium pullulans PL5 involved in the biological control of postharvest pathogens. (United States)

    Zhang, Dianpeng; Spadaro, Davide; Valente, Silvia; Garibaldi, Angelo; Gullino, Maria Lodovica


    An alkaline protease gene was amplified from genomic DNA and cDNA of the antagonistic yeast-like fungus Aureobasidium pullulans PL5, a biocontrol agent effective against Monilinia laxa on stone fruit and Botrytis cinerea and Penicillium expansum on pome fruits. An open reading frame of 1248 bp encoding a 415-amino acid (aa) protein with a calculated molecular weight (M(r)) of 42.9 kDa and an isoelectric point (pI) of 4.5 was characterized. The cDNAALP5 gene had an 18-amino acid signal peptide, one N-gylcosylation, one histidine active site, and one serine active site. The ALP5 gene with a M(r) of 1351 bp contained two introns. One intron was of 54 bp, while the other was of 50 bp. Protein BLAST and phylogenetic tree analysis of the deduced amino sequences from the cDNAALP5 gene showed that the encoded protein had 100% homology to a protease enzyme (ALP2) of a sea strain of A. pullulans, suggesting that the protein ALP5 was an alkaline serine protease. Expression of ALP5 in Escherichia coli BL21 (DE3), followed by identification with Western-blotting, purification with Ni-NTA and analysis of enzymatic activity, yielded an homogeneous recombinant ALP5 which hydrolysed the substrate casein and inhibited the mycelial growth of the pathogens. At its optimal pH of 10.0 and reaction temperature of 50°C, the recombinant protease exhibited the highest activity towards the substrate casein, though the highest stability was at lower temperatures and pH between 7.0 and 9.0. This study provided the direct evidence that extracellular proteases secreted by the antagonist A. pullulans PL5 played a role in the biocontrol activities against some postharvest pathogens of apple and peach. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. Insecticide resistance and intracellular proteases. (United States)

    Wilkins, Richard M


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

  9. Biochemical properties of a novel cysteine protease of Plasmodium vivax, vivapain-4.

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    Byoung-Kuk Na


    Full Text Available Multiple cysteine proteases of malaria parasites are required for maintenance of parasite metabolic homeostasis and egress from the host erythrocyte. In Plasmodium falciparum these proteases appear to mediate the processing of hemoglobin and aspartic proteases (plasmepsins in the acidic food vacuole and the hydrolysis of erythrocyte structural proteins at neutral pH. Two cysteine proteases, vivapain (VX-2 and VX-3 have been characterized in P. vivax, but comprehensive studies of P. vivax cysteine proteases remain elusive.We characterized a novel cysteine protease of P. vivax, VX-4, of which orthologs appears to have evolved differentially in primate plasmodia with strong cladistic affinity toward those of rodent Plasmodium. Recombinant VX-4 demonstrated dual substrate specificity depending on the surrounding micro-environmental pH. Its hydrolyzing activity against benzyloxycarbonyl-Leu-Arg-4-methyl-coumaryl-7-amide (Z-Leu-Arg-MCA and Z-Phe-Arg-MCA was highest at acidic pH (5.5, whereas that against Z-Arg-Arg-MCA was maximal at neutral pH (6.5-7.5. VX-4 preferred positively charged amino acids and Gln at the P1 position, with less strict specificity at P3 and P4. P2 preferences depended on pH (Leu at pH 5.5 and Arg at pH 7.5. Three amino acids that delineate the S2 pocket were substituted in VX-4 compared to VX-2 and VX-3 (Ala90, Gly157 and Glu180. Replacement of Glu180 abolished activity against Z-Arg-Arg-MCA at neutral pH, indicating the importance of this amino acid in the pH-dependent substrate preference. VX-4 was localized in the food vacuoles and cytoplasm of the erythrocytic stage of P. vivax. VX-4 showed maximal activity against actin at neutral pH, and that against P. vivax plasmepsin 4 and hemoglobin was detected at neutral/acidic and acidic pH, respectively.VX-4 demonstrates pH-dependent substrate switching, which might offer an efficient mechanism for the specific cleavage of different substrates in different intracellular

  10. Prevalência de olho seco em portadores de hanseníase de um hospital-colônia em Goiânia Prevalence of dry eye in Hansen's disease patients from a colony hospital in Goiânia, Brazil

    Directory of Open Access Journals (Sweden)

    Karys Carvalho Frazão


    Full Text Available OBJETIVO: Determinar a prevalência de olho seco em hansenianos do Hospital de Dermatologia Sanitária de Goiânia. MÉTODOS: A amostra do presente estudo incluiu 70 hansenianos, do Hospital de Dermatologia Sanitária de Goiânia, e 30 controles, da Fundação Banco de Olhos de Goiás, ambos localizados em Goiânia-GO. Foram realizados exame oftalmológico e testes de Schirmer I, "break-up time" (BUT e rosa bengala em todos estes indivíduos em uma única avaliação. Para o diagnóstico de olho seco foi considerado o teste de Schirmer I menor ou igual a 5 mm e o rosa bengala grau II ou III, em pelo menos um olho. RESULTADOS: Quarenta e quatro (63,0% hansenianos eram do sexo masculino e 22 (73,3% controles, do sexo feminino (p=0,001. A idade média dos hansenianos foi de 61,1±12,5 anos e no grupo controle, 55,7±9,6 anos. Quinze (21,4% hansenianos e quatro (13,3% controles apresentaram diagnóstico de olho seco (p=0,429. A forma virchowiana (HV (74,2% da hanseníase foi a mais prevalente e o olho seco (66,7% foi mais freqüente nesta forma clínica da doença. CONCLUSÃO: A prevalência de olho seco foi maior no grupo de hansenianos, apesar de não ter havido diferença significativa.PURPOSE: To determine the prevalence of dry eye in Hansen's disease patients from the "Hospital de Dermatologia Sanitária de Goiânia". METHODS: The sample of the present study included 70 patients with Hansen's disease, from the "Hospital de Dermatologia Sanitária de Goiânia", and 30 controls, from the "Fundação Banco de Olhos de Goiás", both located in Goiânia-GO, Brazil. Ophthalmologic examination and Schirmer I, break-up time (BUT and rose Bengal staining tests were carried out for all patients in a single evaluation. For the diagnosis of dry eye the Schirmer I equal or less than 5 mm and rose Bengal grade II or III, in at least one eye were considered. RESULTS: Forty-four (63.0% patients with Hansen's disease were male and 22 (73.3% controls were female


    Directory of Open Access Journals (Sweden)

    Olivar José da S. M. Lobato


    Full Text Available

    O presente trabalho foi realizado para informar as pessoas interessadas na estimativa da disponibilidade hídrica no solo do município de Goiânia a municípios limítrofes, pelo Método de C.W. Thornthwaite & Mather (1955, baseado em dados termopluviométricos no período de 1961 a 1971. Apresenta, ainda, a posição geográfíca do município, em relação ao Estado de Goiás. Finalmente, caracteriza o clima do Município de Goiânia, utilizando a classificação de C.W. Thornthwaite 1948, apoiado em duas grandezas que são funções diretas da evapotranspiração potencial: o índice efetivo de umidade e o índice de eficiência térmica. A fórmula climática é a seguinte: B2 W B’4 a’.

    The present work was undertaken to inform interested persons of the estimate of hydric availability in the soil of Goiânia county and surrounding counties. The method used was C.W. Thornthwaite and Mather (1955, based on thermo-pluviometrics within the period 1961—1971. The geographic position of the county, in relation to the State of Goiás, is also presented. Finally the climate of Goiânia County, is described using Thornthwaite's 1948 classification.

  12. Governing Civil Service Pay in China, by Alfred M. Wu. Copenhagen: NIAS Press, 2014. xvi+236 pp

    DEFF Research Database (Denmark)

    Brødsgaard, Kjeld Erik


    Book review of: Governing Civil Service Pay in China by Alfred M. Wu. Copenhagen: NIAS Press, 2014.......Book review of: Governing Civil Service Pay in China by Alfred M. Wu. Copenhagen: NIAS Press, 2014....

  13. In vitro protease cleavage and computer simulations reveal the HIV-1 capsid maturation pathway (United States)

    Ning, Jiying; Erdemci-Tandogan, Gonca; Yufenyuy, Ernest L.; Wagner, Jef; Himes, Benjamin A.; Zhao, Gongpu; Aiken, Christopher; Zandi, Roya; Zhang, Peijun


    HIV-1 virions assemble as immature particles containing Gag polyproteins that are processed by the viral protease into individual components, resulting in the formation of mature infectious particles. There are two competing models for the process of forming the mature HIV-1 core: the disassembly and de novo reassembly model and the non-diffusional displacive model. To study the maturation pathway, we simulate HIV-1 maturation in vitro by digesting immature particles and assembled virus-like particles with recombinant HIV-1 protease and monitor the process with biochemical assays and cryoEM structural analysis in parallel. Processing of Gag in vitro is accurate and efficient and results in both soluble capsid protein and conical or tubular capsid assemblies, seemingly converted from immature Gag particles. Computer simulations further reveal probable assembly pathways of HIV-1 capsid formation. Combining the experimental data and computer simulations, our results suggest a sequential combination of both displacive and disassembly/reassembly processes for HIV-1 maturation.

  14. Las ni??as, las ??ltimas. Un estudio sobre el abandono escolar femenino en Kibosho Central (Tanzania)


    John, Felisa; Francisco, Andrea; Moliner Miravet, Lid??n


    Uno de los Objetivos del Milenio para 2015 era lograr el acceso a la ense??anza primaria universal para todos y todas pero a??n hay 58 millones de ni??os y ni??as de entre 6 y 11 a??os sin escolarizar, una situaci??n que afecta sobre todo a las ni??as. Las tasas m??s bajas de escolarizaci??n primaria corresponden a ??frica subsahariana, donde s??lo el 65% de los ni??os y ni??as en edad de estudiar acuden a la escuela (UNESCO, 2014). A estas cifras, adem??s, hay que a??adir que una...

  15. Proteolytic crosstalk in multi-protease networks (United States)

    Ogle, Curtis T.; Mather, William H.


    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.

  16. Tri-domain Bifunctional Inhibitor of Metallocarboxypeptidases A and Serine Proteases Isolated from Marine Annelid Sabellastarte magnifica* (United States)

    Alonso-del-Rivero, Maday; Trejo, Sebastian A.; Reytor, Mey L.; Rodriguez-de-la-Vega, Monica; Delfin, Julieta; Diaz, Joaquin; González-González, Yamile; Canals, Francesc; Chavez, Maria Angeles; Aviles, Francesc X.


    This study describes a novel bifunctional metallocarboxypeptidase and serine protease inhibitor (SmCI) isolated from the tentacle crown of the annelid Sabellastarte magnifica. SmCI is a 165-residue glycoprotein with a molecular mass of 19.69 kDa (mass spectrometry) and 18 cysteine residues forming nine disulfide bonds. Its cDNA was cloned and sequenced by RT-PCR and nested PCR using degenerated oligonucleotides. Employing this information along with data derived from automatic Edman degradation of peptide fragments, the SmCI sequence was fully characterized, indicating the presence of three bovine pancreatic trypsin inhibitor/Kunitz domains and its high homology with other Kunitz serine protease inhibitors. Enzyme kinetics and structural analyses revealed SmCI to be an inhibitor of human and bovine pancreatic metallocarboxypeptidases of the A-type (but not B-type), with nanomolar Ki values. SmCI is also capable of inhibiting bovine pancreatic trypsin, chymotrypsin, and porcine pancreatic elastase in varying measures. When the inhibitor and its nonglycosylated form (SmCI N23A mutant) were overproduced recombinantly in a Pichia pastoris system, they displayed the dual inhibitory properties of the natural form. Similarly, two bi-domain forms of the inhibitor (recombinant rSmCI D1-D2 and rSmCI D2-D3) as well as its C-terminal domain (rSmCI-D3) were also overproduced. Of these fragments, only the rSmCI D1-D2 bi-domain retained inhibition of metallocarboxypeptidase A but only partially, indicating that the whole tri-domain structure is required for such capability in full. SmCI is the first proteinaceous inhibitor of metallocarboxypeptidases able to act as well on another mechanistic class of proteases (serine-type) and is the first of this kind identified in nature. PMID:22411994

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

    Sharma, Ranu; Suresh, C G


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

  18. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    International Nuclear Information System (INIS)

    Nishikado, Hideto; Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko; Ogawa, Hideoki; Okumura, Ko; Takai, Toshiro


    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity

  19. Cysteine protease antigens cleave CD123, the α subunit of murine IL-3 receptor, on basophils and suppress IL-3-mediated basophil expansion

    Energy Technology Data Exchange (ETDEWEB)

    Nishikado, Hideto [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Fujimura, Tsutomu; Taka, Hikari; Mineki, Reiko [Laboratory of Proteomics and Biomolecular Science, BioMedical Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Ogawa, Hideoki; Okumura, Ko [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan); Takai, Toshiro, E-mail: [Atopy (Allergy) Research Center, Juntendo University Graduate School of Medicine, Tokyo (Japan)


    Th2 type immune responses are essential for protective immunity against parasites and play crucial roles in allergic disorders. Helminth parasites secrete a variety of proteases for their infectious cycles including for host entry, tissue migration, and suppression of host immune effector cell function. Furthermore, a number of pathogen-derived antigens, as well as allergens such as papain, belong to the family of cysteine proteases. Although the link between protease activity and Th2 type immunity is well documented, the mechanisms by which proteases regulate host immune responses are largely unknown. Here, we demonstrate that the cysteine proteases papain and bromelain selectively cleave the α subunit of the IL-3 receptor (IL-3Rα/CD123) on the surface of murine basophils. The decrease in CD123 expression on the cell surface, and the degradation of the extracellular domain of recombinant CD123 were dependent on the protease activity of papain and bromelain. Pre-treatment of murine basophils with papain resulted in inhibition of IL-3-IL-3R signaling and suppressed IL-3- but not thymic stromal lymphopoietin-induced expansion of basophils in vitro. Our unexpected findings illuminate a novel mechanism for the regulation of basophil functions by protease antigens. Because IL-3 plays pivotal roles in the activation and proliferation of basophils and in protective immunity against helminth parasites, pathogen-derived proteases might contribute to the pathogenesis of infections by regulating IL-3-mediated functions in basophils. - Highlights: • We identified the murine IL3R as a novel target of papain-family cysteine proteases. • Papain-family cysteine proteases cleaved IL3Rα/CD123 on murine basophils. • Papain suppressed IL3- but not TSLP-induced expansion of murine basophils. • The inactivation of IL3R might be a strategy for pathogens to suppress host immunity.

  20. Evidence that two ATP-dependent (Lon proteases in Borrelia burgdorferi serve different functions.

    Directory of Open Access Journals (Sweden)

    James L Coleman


    Full Text Available The canonical ATP-dependent protease Lon participates in an assortment of biological processes in bacteria, including the catalysis of damaged or senescent proteins and short-lived regulatory proteins. Borrelia spirochetes are unusual in that they code for two putative ATP-dependent Lon homologs, Lon-1 and Lon-2. Borrelia burgdorferi, the etiologic agent of Lyme disease, is transmitted through the blood feeding of Ixodes ticks. Previous work in our laboratory reported that B. burgdorferi lon-1 is upregulated transcriptionally by exposure to blood in vitro, while lon-2 is not. Because blood induction of Lon-1 may be of importance in the regulation of virulence factors critical for spirochete transmission, the clarification of functional roles for these two proteases in B. burgdorferi was the object of this study. On the chromosome, lon-2 is immediately downstream of ATP-dependent proteases clpP and clpX, an arrangement identical to that of lon of Escherichia coli. Phylogenetic analysis revealed that Lon-1 and Lon-2 cluster separately due to differences in the NH(2-terminal substrate binding domains that may reflect differences in substrate specificity. Recombinant Lon-1 manifested properties of an ATP-dependent chaperone-protease in vitro but did not complement an E. coli Lon mutant, while Lon-2 corrected two characteristic Lon-mutant phenotypes. We conclude that B. burgdorferi Lons -1 and -2 have distinct functional roles. Lon-2 functions in a manner consistent with canonical Lon, engaged in cellular homeostasis. Lon-1, by virtue of its blood induction, and as a unique feature of the Borreliae, may be important in host adaptation from the arthropod to a warm-blooded host.

  1. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

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


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

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

    Directory of Open Access Journals (Sweden)

    Ming-Yang Zhou

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

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

    International Nuclear Information System (INIS)

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


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  5. Diversity of the land resources in the Amazonian State of Rondônia, Brazil Diversidade dos terrenos e solos do estado de Rondônia, Brasil

    Directory of Open Access Journals (Sweden)

    T.T. Cochrane


    Full Text Available Conflicting opinions are recorded in the literature concerning the suitability of Amazon lands for sustainable agriculture following deforestation. This article has been written to shed light on this question by summarizing climate, landform, soil and vegetation features from the findings of a land resource study of the Brazilian state of Rondônia in south-west Amazonia. The work, which followed the World Soils and Terrain Digital Database (SOTER methodology, was financed by the World Bank. During the course of the survey special emphasis was given to studying soils; 2914 profiles were analyzed and recorded. The study identified a complex pattern of land units with clear differences in climate, landform, soils and native vegetation. Forested areas mosaic with lesser areas of natural savannas. The latter occur on both poorly-drained and well-drained, albeit nutrient deficient sandy soils. The tallest and most vigorous forests or their remnants were seen growing on well-drained soils formed from nutrient-rich parent materials. Many of these soils could, or are being used for productive agriculture. Soils developed on nutrient-poor parent materials support forests that are significantly lower in height, and would require large lime and fertilizer inputs for agriculture. Low forests with high palm populations and minor areas of wet land savannas cover the poorly drained soils. It is evident that forest clearing in the past was indiscriminant; this cannot be condoned. The diversity of land conditions found throughout Rondônia would suggest that many past studies in the Amazon have simply been too broad to identify significant soil differences.Opiniões divergentes são encontradas na literatura com respeito ao uso de terras da Amazônia para agricultura sustentável apos desmatamento. Este artigo busca clarificar a questão com um resumo dos resultados de um estudo dos recursos naturais (clima, atributos de solos e terrenos, e vegetação do estado

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

    DEFF Research Database (Denmark)

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


    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...

  7. Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2. (United States)

    Sheffield, William P; Eltringham-Smith, Louise J; Bhakta, Varsha


    The Kunitz Protease Inhibitor (KPI) domain of protease nexin 2 (PN2) potently inhibits coagulation factor XIa. Recombinant KPI has been shown to inhibit thrombosis in mouse models, but its clearance from the murine circulation remains uncharacterized. The present study explored the pharmacokinetic and pharmacodynamic effects of fusing KPI to human serum albumin (HSA) in fusion protein KPIHSA. Hexahistidine-tagged KPI (63 amino acids) and KPIHSA (656 amino acids) were expressed in Pichia pastoris yeast and purified by nickel-chelate chromatography. Clearance profiles in mice were determined, as well as the effects of KPI or KPIHSA administration on FeCl3-induced vena cava thrombus size or carotid artery time to occlusion, respectively. Fusion to HSA increased the mean terminal half-life of KPI by 8-fold and eliminated its interaction with the low density lipoprotein receptor-related protein. KPI and KPIHSA similarly reduced thrombus size and occlusion in both venous and arterial thrombosis models when administered at the time of injury, but only KPI was effective when administered one hour before injury. Albumin fusion deflects KPI from rapid in vivo clearance without impairing its antithrombotic properties and widens its potential therapeutic window. © 2018 The Author(s). Published by S. Karger AG, Basel.

  8. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

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

  9. A biotechnology perspective of fungal proteases

    Directory of Open Access Journals (Sweden)

    Paula Monteiro de Souza


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

  10. Ainda a Amazônia

    Directory of Open Access Journals (Sweden)

    Mario B. Aragão


    Full Text Available No momento há grande preocupação com tentativas de internacionalizar a Amazônia. Entretanto, um exame rápido da história da região mostra que a reação, provocada por tentativas semelhantes, sempre deu resultados benéficos. O problema mais sério que o desmatamento da Amazônia provocará, é a mudança no total e, possivelmente, no regime das chuvas, tanto na região como nas áreas vizinhas. Os 6,5 trilhões de metros cúbicos de água que são evaporados, anualmente, pela floresta, em sua quase totalidade deixarão de entrar na circulação atmosférica. Essa quantidade de vapor d'água é que fornece 50% da chuva que cai na bacia. A questão do "pulmão do mundo" já foi suficientemente explicada, pois, uma floresta em estado "climax" não pode ter saldo de oxigênio. Além disso, o grande lançador de gás carbônico na atmosfera é o consumismo dos países ricos. Entretanto, resultados recentes parecem mostrar que esse gás carbônico não está provocando efeito estufa e aumentando a tempertura da terra. A experiência internacional e também a nacional têm mostrado que a melhor maneira de proteger uma floresta é dar a ela uma utilidade, logicamente, do ponto de vista do homem. A melhor proposta surgida até hoje foi a da criação das florestas regionais de rendimento, onde empresas madeireiras receberiam concessão para explorá-las, sem se tornarem proprietárias nem da terra nem da vegetação. Não é de hoje a vinculação entre floresta amazônica e dívida externa. Desde os projetos pecuários até os grandes empreendimentos industriais e de mineração, todos têm sido incentivados na ilusão de obter divisas para o pagamento da dívida externa. O problema das represas precisa ser repensado; está sendo inutilizada muita terra para a produção de pouca energia que, além do mais, pouco beneficiará a população brasileira. Os garimpos, tradicionalmente, têm sustentado o contrabando, principalmente de ouro e as

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

    International Nuclear Information System (INIS)

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


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


    Directory of Open Access Journals (Sweden)

    Edithe Pereira


    Full Text Available RESUMO: O artigo apresenta os problemas decorrentes da prática do turismo sem controle que vem acontecendo em sítios arqueológicos na Amazônia. Apresenta-se a situação atual de alguns sítios localizados em três regiões distintas no Pará e propõem-se recomendações básicas para uso turístico de sítios arqueológicos. PALAVRAS-CHAVE: Patrimônio arqueológico; turismo; Amazônia.ABSTRACT:This article discusses the main problems that have affected Amazonian archaeological sites as a consequence of unsupervised tourist practices. The current state of sites located in three different regions of Pará are examined, while some basic procedures for the touristic access to archaeological sites are recommended.KEY-WORKS: Archaeological heritage; tourism; Amazon Region.

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  14. Recombinant Passenger Proteins Can Be Conveniently Purified by One-Step Affinity Chromatography. (United States)

    Wang, Hua-zhen; Chu, Zhi-zhan; Chen, Chang-chao; Cao, Ao-cheng; Tong, Xin; Ouyang, Can-bin; Yuan, Qi-hang; Wang, Mi-nan; Wu, Zhong-kun; Wang, Hai-hong; Wang, Sheng-bin


    Fusion tag is one of the best available tools to date for enhancement of the solubility or improvement of the expression level of recombinant proteins in Escherichia coli. Typically, two consecutive affinity purification steps are often necessitated for the purification of passenger proteins. As a fusion tag, acyl carrier protein (ACP) could greatly increase the soluble expression level of Glucokinase (GlcK), α-Amylase (Amy) and GFP. When fusion protein ACP-G2-GlcK-Histag and ACP-G2-Amy-Histag, in which a protease TEV recognition site was inserted between the fusion tag and passenger protein, were coexpressed with protease TEV respectively in E. coli, the efficient intracellular processing of fusion proteins was achieved. The resulting passenger protein GlcK-Histag and Amy-Histag accumulated predominantly in a soluble form, and could be conveniently purified by one-step Ni-chelating chromatography. However, the fusion protein ACP-GFP-Histag was processed incompletely by the protease TEV coexpressed in vivo, and a large portion of the resulting target protein GFP-Histag aggregated in insoluble form, indicating that the intracellular processing may affect the solubility of cleaved passenger protein. In this context, the soluble fusion protein ACP-GFP-Histag, contained in the supernatant of E. coli cell lysate, was directly subjected to cleavage in vitro by mixing it with the clarified cell lysate of E. coli overexpressing protease TEV. Consequently, the resulting target protein GFP-Histag could accumulate predominantly in a soluble form, and be purified conveniently by one-step Ni-chelating chromatography. The approaches presented here greatly simplify the purification process of passenger proteins, and eliminate the use of large amounts of pure site-specific proteases.

  15. Os fatores genéticos da insônia - Uma revisão de literatura

    Directory of Open Access Journals (Sweden)

    Guilherme Sousa Ferreira


    Full Text Available A insônia é um dos distúrbios do sono caracterizado pela dificuldade em iniciar ou manter o sono. Isso se reflete em um sono de baixa qualidade que pode causar problemas ao longo do dia, como cansaço e falta de energia. Sob essa condição, estão associados alguns fatores genéticos, entre eles os genes ABCC9, 5-HTT e Clock. A presente revisão de literatura tem por objetivo a explanação acerca dos fatores genéticos relacionados com o distúrbio da insônia. Trata-se de uma revisão de literatura de forma sistemática, realizada por meio de um levantamento bibliográfico que procura reunir os conhecimentos sobre as características da insônia e sua herança gênica. Encontrou-se uma relevância de achados autorais valiosos para a síntese dessa pesquisa. Portanto, a partir do estudo abordado nessa revisão, torna-se evidente a importância do conhecimento das causas genéticas do distúrbio da insônia para a melhor abordagem clínica, haja vista a relevância desse mecanismo biológico.

  16. Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin. (United States)

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


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

  17. Advances in protease engineering for laundry detergents. (United States)

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


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

  18. Estabelecimento de leguminosas arbóreas em pastos de capim-marandu e tanzânia Establishment of leguminous trees in marandu and tanzânia pastures

    Directory of Open Access Journals (Sweden)

    Paulo Francisco Dias


    Full Text Available O objetivo deste trabalho foi avaliar o desempenho de mudas sem proteção (de cercas ou estacas de quatro espécies de leguminosas arbóreas e uma mistura eqüitativa dessas espécies, introduzidas em pastagens de Brachiaria brizantha cv. Marandu e Panicum maximum cv. Tanzânia, na presença de gado. O delineamento experimental foi o inteiramente ao acaso, em esquema fatorial 2x5, duas gramíneas (marandu e tanzânia e quatro espécies de leguminosas (Mimosa artemisiana, Pseudosamanea guachapele, Enterolobium contortisiliquum, Acacia farnesiana e uma mistura dessas espécies, com três repetições. Avaliaram-se: altura da muda, diâmetro do caule, diâmetro da copa, sobrevivência da muda, freqüência de pastejo e ocorrência de formigas. As diferenças estatísticas entre as médias da variável canônica principal, pelo teste de Scott-Knott, indicaram a formação de três agrupamentos, tendo-se destacado o grupo formado pelos tratamentos M. artemisiana e mistura de leguminosas, nos dois pastos, mais E. contortisiliquum e A. farnesiana, nos pastos dos capins marandu e tanzânia, respectivamente. Diferenças entre as médias dos tratamentos relativas a cada variável, calculadas por meio de intervalos de confiança de Bonferroni, mostraram que mudas de M. artemisiana apresentaram maior altura e sobrevivência em pasto de capim-marandu. Mudas dessa leguminosa, sem proteção, são indicadas para ser introduzidas, nas pastagens de capim-marandu da região, na presença do gado.The objective of this work was to evaluate the performance of nonprotected (by fences or pickets seedlings of leguminous tree species and an equitable mix of these species, introduced in pastures of Brachiaria brizantha cv. Marandu and Panicum maximum cv. Tanzânia, in the presence of cattle. The experimental design was a completely randomized one, in a 2x5 factorial arrangement, with two grasses (marandu and tanzânia and four leguminous species (Mimosa artemisiana

  19. O experimento de grande escala da biosfera-atmosfera na Amazônia (LBA); plano experimental conciso

    NARCIS (Netherlands)

    LBAScience Planning Group (Cachoeira Paulista),


    O experimento de grande escala da biosfera atmosfera na Amazónia (LBA) tem como objectivo aperfeiçoar o conhecimento do funcionamento climatológico, ecológico, biogeoquímico e hidrológico da Amazónia. Isto chamará a atençao para os efeitos das alteraçoes da ocupaçao do solo e clima, nestas funçoes,

  20. Pessoa em Bethânia: os versos do desassossego na voz do encantamento


    Barros, Andre Luiz Calsone


    Pessoa em Bethânia tem por tema a recriação dos versos de Caeiro no espetáculo Rosa dos Ventos O Show Encantado, tendo por intérprete Maria Bethânia. O corpus é o Poema VIII de Alberto Caeiro, heterônimo de Fernando Pessoa, da obra O Guardador de Rebanhos (1911 1912), transformado em roteiro dramáticomusical e tornado performance no espetáculo Rosa dos Ventos. Reinterpretado por meio da voz, do corpo, da música e dos mais variados recursos cênicos, o poema de Fernando ...

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

    Directory of Open Access Journals (Sweden)

    Jeong Hee Kim

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

  2. Understanding serine proteases implications on Leishmania spp lifecycle. (United States)

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


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

  3. Isolation, cDNA cloning, and structure-based functional characterization of oryctin, a hemolymph protein from the coconut rhinoceros beetle, Oryctes rhinoceros, as a novel serine protease inhibitor. (United States)

    Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru


    We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant (13)C,(15)N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with K(i) values of 3.9 × 10(-10) m, 6.2 × 10(-10) m, 1.4 × 10(-9) m, and 1.2 × 10(-8) m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections.

  4. A Novel Recombinant DNA System for High Efficiency Affinity Purification of Proteins in Saccharomyces cerevisiae

    Directory of Open Access Journals (Sweden)

    Brian H. Carrick


    Full Text Available Isolation of endogenous proteins from Saccharomyces cerevisiae has been facilitated by inserting encoding polypeptide affinity tags at the C-termini of chromosomal open reading frames (ORFs using homologous recombination of DNA fragments. Tagged protein isolation is limited by a number of factors, including high cost of affinity resins for bulk isolation and low concentration of ligands on the resin surface, leading to low isolation efficiencies and trapping of contaminants. To address this, we have created a recombinant “CelTag” DNA construct from which PCR fragments can be created to easily tag C-termini of S. cerevisiae ORFs using selection for a nat1 marker. The tag has a C-terminal cellulose binding module to be used in the first affinity step. Microgranular cellulose is very inexpensive and has an effectively continuous ligand on its surface, allowing rapid, highly efficient purification with minimal background. Cellulose-bound proteins are released by specific cleavage of an included site for TEV protease, giving nearly pure product. The tag can be lifted from the recombinant DNA construct either with or without a 13x myc epitope tag between the target ORF and the TEV protease site. Binding of CelTag protein fusions to cellulose is stable to high salt, nonionic detergents, and 1 M urea, allowing stringent washing conditions to remove loosely associated components, as needed, before specific elution. It is anticipated that this reagent could allow isolation of protein complexes from large quantities of yeast extract, including soluble, membrane-bound, or nucleic acid-associated assemblies.

  5. Gender inequality: Behind maternal mortality in Nias Island, North Sumatra, Indonesia: Towards a gender audit


    Zaluchu, F.


    Singling out Nias Island in North Sumatra Indonesia as the research location, this research attempts to analyze local aspects pertaining to gender inequality in the context of maternal health in Indonesia. This research aims to portray the presence of gender inequality in Nias, and how it affects the reproductive health of and poses Niasan women to the risk of maternal death. It also carefully examines the social situations in which gender inequality is established and reproduced by the exist...

  6. A genomic survey of proteases in Aspergilli

    NARCIS (Netherlands)

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


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

  7. Avaliação clínica e manejo da insônia em pacientes pediátricos


    Nunes,Magda Lahorgue; Cavalcante,Verônica


    OBJETIVOS: O objetivo deste artigo é revisar as características clínicas, a investigação e o tratamento da insônia na faixa etária pediátrica. FONTE DOS DADOS: Foi realizada pesquisa na MEDLINE, nos últimos 5 anos (1999-2004), utilizando os unitermos distúrbios do sono, insônia e infância. Foram incluídos artigos e livros textos clássicos sobre o tema, sendo elaborado um roteiro crítico para avaliação e manejo da insônia em pacientes pediátricos. SÍNTESE DOS DADOS: O artigo foi estruturado em...

  8. Cytomegalovirus protease targeted prodrug development. (United States)

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


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

  9. An overview on molecular chaperones enhancing solubility of expressed recombinant proteins with correct folding. (United States)

    Mamipour, Mina; Yousefi, Mohammadreza; Hasanzadeh, Mohammad


    The majority of research topics declared that most of the recombinant proteins have been expressed by Escherichia coli in basic investigations. But the majority of high expressed proteins formed as inactive recombinant proteins that are called inclusion body. To overcome this problem, several methods have been used including suitable promoter, environmental factors, ladder tag to secretion of proteins into the periplasm, gene protein optimization, chemical chaperones and molecular chaperones sets. Co-expression of the interest protein with molecular chaperones is one of the common methods The chaperones are a group of proteins, which are involved in making correct folding of recombinant proteins. Chaperones are divided two groups including; cytoplasmic and periplasmic chaperones. Moreover, periplasmic chaperones and proteases can be manipulated to increase the yields of secreted proteins. In this article, we attempted to review cytoplasmic chaperones such as Hsp families and periplasmic chaperones including; generic chaperones, specialized chaperones, PPIases, and proteins involved in disulfide bond formation. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Hepatitis C Virus Genotype 1 to 6 Protease Inhibitor Escape Variants: In Vitro Selection, Fitness, and Resistance Patterns in the Context of the Infectious Viral Life Cycle. (United States)

    Serre, Stéphanie B N; Jensen, Sanne B; Ghanem, Lubna; Humes, Daryl G; Ramirez, Santseharay; Li, Yi-Ping; Krarup, Henrik; Bukh, Jens; Gottwein, Judith M


    Hepatitis C virus (HCV) NS3 protease inhibitors (PIs) are important components of novel HCV therapy regimens. Studies of PI resistance initially focused on genotype 1. Therefore, knowledge about the determinants of PI resistance for the highly prevalent genotypes 2 to 6 remains limited. Using Huh7.5 cell culture-infectious HCV recombinants with genotype 1 to 6 NS3 protease, we identified protease positions 54, 155, and 156 as hot spots for the selection of resistance substitutions under treatment with the first licensed PIs, telaprevir and boceprevir. Treatment of a genotype 2 isolate with the newer PIs vaniprevir, faldaprevir, simeprevir, grazoprevir, paritaprevir, and deldeprevir identified positions 156 and 168 as hot spots for resistance; the Y56H substitution emerged for three newer PIs. Substitution selection also depended on the specific recombinant. The substitutions identified conferred cross-resistance to several PIs; however, most substitutions selected under telaprevir or boceprevir treatment conferred less resistance to certain newer PIs. In a single-cycle production assay, across genotypes, PI treatment primarily decreased viral replication, which was rescued by PI resistance substitutions. The substitutions identified resulted in differential effects on viral fitness, depending on the original recombinant and the substitution. Across genotypes, fitness impairment induced by resistance substitutions was due primarily to decreased replication. Most combinations of substitutions that were identified increased resistance or fitness. Combinations of resistance substitutions with fitness-compensating substitutions either rescued replication or compensated for decreased replication by increasing assembly. This comprehensive study provides insight into the selection patterns and effects of PI resistance substitutions for HCV genotypes 1 to 6 in the context of the infectious viral life cycle, which is of interest for clinical and virological HCV research

  11. Role of Proteases in Chronic Obstructive Pulmonary Disease

    Directory of Open Access Journals (Sweden)

    Kailash C. Pandey


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

  12. Insônia na menopausa e perimenopausa: características clínicas e opções terapêuticas


    Soares,Claudio N.


    Transtornos do sono e insônia são mais freqüentemente observados entre as mulheres do que entre os homens durante a vida adulta. Entre outros fatores, especula-se que alta comorbidade psiquiátrica para transtornos afetivos e ansiosos entre as mulheres contribua para maior ocorrência de insônia. Além disso, períodos de intensa variabilidade hormonal (por exemplo, gestação e puerpério, perimenopausa) parecem estar relacionados com maior incidência de insônia e de transtornos respiratórios do so...

  13. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)



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

  14. Mosaic serine proteases in the mammalian central nervous system. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Madhusudhan Budatha

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

  16. Extracellular proteases of Trichoderma species. A review. (United States)

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


    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.

  17. Antiviral cationic peptides as a strategy for innovation in global health therapeutics for dengue virus: high yield production of the biologically active recombinant plectasin peptide. (United States)

    Rothan, Hussin A; Mohamed, Zulqarnain; Suhaeb, Abdulrazzaq M; Rahman, Noorsaadah Abd; Yusof, Rohana


    Dengue virus infects millions of people worldwide, and there is no vaccine or anti-dengue therapeutic available. Antimicrobial peptides have been shown to possess effective antiviral activity against various viruses. One of the main limitations of developing these peptides as potent antiviral drugs is the high cost of production. In this study, high yield production of biologically active plectasin peptide was inexpensively achieved by producing tandem plectasin peptides as inclusion bodies in E. coli. Antiviral activity of the recombinant peptide towards dengue serotype-2 NS2B-NS3 protease (DENV2 NS2B-NS3pro) was assessed as a target to inhibit dengue virus replication in Vero cells. Single units of recombinant plectasin were collected after applying consecutive steps of refolding, cleaving by Factor Xa, and nickel column purification to obtain recombinant proteins of high purity. The maximal nontoxic dose (MNTD) of the recombinant peptide against Vero cells was 20 μM (100 μg/mL). The reaction velocity of DENV2 NS2B-NS3pro decreased significantly after increasing concentrations of recombinant plectasin were applied to the reaction mixture. Plectasin peptide noncompetitively inhibited DENV2 NS2B-NS3pro at Ki value of 5.03 ± 0.98 μM. The percentage of viral inhibition was more than 80% at the MNTD value of plectasin. In this study, biologically active recombinant plectasin which was able to inhibit dengue protease and viral replication in Vero cells was successfully produced in E. coli in a time- and cost- effective method. These findings are potentially important in the development of potent therapeutics against dengue infection.

  18. The story of an exceptional serine protease, tissue-type plasminogen activator (tPA). (United States)

    Hébert, M; Lesept, F; Vivien, D; Macrez, R


    The only acute treatment of ischemic stroke approved by the health authorities is tissue recombinant plasminogen activator (tPA)-induced thrombolysis. Under physiological conditions, tPA, belonging to the serine protease family, is secreted by endothelial and brain cells (neurons, astrocytes, microglia, oligodendrocytes). Although revascularisation induced by tPA is beneficial during a stroke, research over the past 20 years shows that tPA can also be deleterious for the brain parenchyma. Thus, in this review of the literature, after a brief history on the discovery of tPA, we reviewed current knowledge of mechanisms by which tPA can influence brain function in physiological and pathological conditions. Copyright © 2015 Elsevier Masson SAS. All rights reserved.

  19. Curcumin derivatives as HIV-1 protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

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


    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.

  20. Novel HIV-1 recombinants spreading across multiple risk groups in the United Kingdom: the identification and phylogeography of Circulating Recombinant Form (CRF 50_A1D.

    Directory of Open Access Journals (Sweden)

    Geraldine M Foster

    Full Text Available An increase in non-B HIV-1 infections among men who have sex with men (MSM in the United Kingdom (UK has created opportunities for novel recombinants to arise and become established. We used molecular mapping to characterize the importance of such recombinants to the UK HIV epidemic, in order to gain insights into transmission dynamics that can inform control strategies.A total of 55,556 pol (reverse transcriptase and protease sequences in the UK HIV Drug Resistance Database were analyzed using Subtype Classification Using Evolutionary Algorithms (SCUEAL. Overall 72 patients shared the same A1/D recombination breakpoint in pol, comprising predominantly MSM but also heterosexuals and injecting drug users (IDUs. In six MSM, full-length single genome amplification of plasma HIV-1 RNA was performed in order to characterize the A1/D recombinant. Subtypes and recombination breakpoints were identified using sliding window and jumping profile hidden markov model approaches. Global maximum likelihood trees of gag, pol and env genes were drawn using FastTree version 2.1. Five of the six strains showed the same novel A1/D recombinant (8 breakpoints, which has been classified as CRF50_A1D. The sixth strain showed a complex CRF50_A1D/B/U structure. Divergence dates and phylogeographic inferences were determined using Bayesian Evolutionary Analysis using Sampling Trees (BEAST. This estimated that CRF50_A1D emerged in the UK around 1992 in MSM, with subsequent transmissions to heterosexuals and IDUs. Analysis of CRF50_A1D/B/U demonstrated that around the year 2000 CRF50_A1D underwent recombination with a subtype B strain.We report the identification of CRF50_A1D, a novel circulating recombinant that emerged in UK MSM around 1992, with subsequent onward transmission to heterosexuals and IDUs, and more recent recombination with subtype B. These findings highlight the changing dynamics of HIV transmission in the UK and the converging of the two previously

  1. Receptores do factor estimulante de colónias de macrófagos do robalo


    Oliveira, Márcio Adriano Guiomar de


    O receptor do factor estimulante de colónias de macrófagos, também conhecido como receptor do factor estimulante de colónias-1 (CSF1R), é um receptor de um factor de crescimento hematopoiético que é especificamente expresso em células do sistema fagocítico-mononuclear e desempenha um papel essencial no desenvolvimento e regulação destas células. O CSF1R já foi descrito em vários mamíferos e a sua biologia tem sido exaustivamente caracterizada nestes vertebrados mas o conheci...

  2. Heparin-binding peptide as a novel affinity tag for purification of recombinant proteins. (United States)

    Morris, Jacqueline; Jayanthi, Srinivas; Langston, Rebekah; Daily, Anna; Kight, Alicia; McNabb, David S; Henry, Ralph; Kumar, Thallapuranam Krishnaswamy Suresh


    Purification of recombinant proteins constitutes a significant part of the downstream processing in biopharmaceutical industries. Major costs involved in the production of bio-therapeutics mainly depend on the number of purification steps used during the downstream process. Affinity chromatography is a widely used method for the purification of recombinant proteins expressed in different expression host platforms. Recombinant protein purification is achieved by fusing appropriate affinity tags to either N- or C- terminus of the target recombinant proteins. Currently available protein/peptide affinity tags have proved quite useful in the purification of recombinant proteins. However, these affinity tags suffer from specific limitations in their use under different conditions of purification. In this study, we have designed a novel 34-amino acid heparin-binding affinity tag (HB-tag) for the purification of recombinant proteins expressed in Escherichia coli (E. coli) cells. HB-tag fused recombinant proteins were overexpressed in E. coli in high yields. A one-step heparin-Sepharose-based affinity chromatography protocol was developed to purify HB-fused recombinant proteins to homogeneity using a simple sodium chloride step gradient elution. The HB-tag has also been shown to facilitate the purification of target recombinant proteins from their 8 M urea denatured state(s). The HB-tag has been demonstrated to be successfully released from the fusion protein by an appropriate protease treatment to obtain the recombinant target protein(s) in high yields. Results of the two-dimensional NMR spectroscopy experiments indicate that the purified recombinant target protein(s) exist in the native conformation. Polyclonal antibodies raised against the HB-peptide sequence, exhibited high binding specificity and sensitivity to the HB-fused recombinant proteins (∼10 ng) in different crude cell extracts obtained from diverse expression hosts. In our opinion, the HB-tag provides a

  3. Recombinant human adenovirus-5 expressing capsid proteins of Indian vaccine strains of foot-and-mouth disease virus elicits effective antibody response in cattle (United States)

    Recombinant adenovirus-5 vectored foot-and-mouth disease constructs (Ad5- FMD) were made for three Indian vaccine virus serotypes O,A and Asia 1. Constructs co-expressing foot-and- mouth disease virus (FMDV) capsid and viral 3C protease sequences, were evaluated for their ability to induce a neutral...

  4. Insônia primária: diagnóstico diferencial e tratamento Primary insomnia: differential diagnosis and treatment

    Directory of Open Access Journals (Sweden)

    Jaime M Monti


    Full Text Available A insônia primária é uma dissonia caracterizada pela dificuldade em iniciar e/ou manter o sono e pela sensação de não ter um sono reparador durante um período não inferior a 1 mês. Do ponto de vista polissonográfico, é acompanhada de alterações na indução, na continuidade e na estrutura do sono. Geralmente aparece no adulto jovem, é mais freqüente na mulher e tem um desenvolvimento crônico. A insônia primária é observada de 12,5% a 22,2% dos pacientes portadores de insônia crônica, sendo precedida em freqüência somente na insônia de depressão maior. A insônia primária crônica deve se diferenciar da insônia vinculada a uma higiene inadequada do sono, uma síndrome depressiva ou um transtorno de ansiedade generalizado. O tratamento da insônia primária inclui: higiene adequada do sono, terapia cognitiva e de conduta e uso de fármacos hipnóticos. Entre esses últimos, se destacam o zolpidem e a zopiclona, que melhoram significativamente o sono sem alterar sua estrutura ou induzir a uma reincidência da insônia logo após uma interrupção brusca. Além disso, o desenvolvimento de fármaco-dependência e de vício é muito pouco freqüente.Primary insomnia is a dyssomnia characterized by a complaint of difficulty in initiating or maintaining sleep and the absence of restorative sleep that lasts for at least 1 month. The polysomnographic test shows alterations in the induction, continuity and structure of sleep. Primary insomnia typically begins in young adulthood, has a chronic course, and it is more prevalent among women. Its prevalence among patients with chronic insomnia ranges from 12.5% to 22.2%. Primary insomnia must be distinguished from insomnia related to inadequate sleep hygiene or another mental disorder, such as generalized anxiety disorder or a mood disorder. The treatment of primary insomnia consists of nonpharmacological strategies (sleep hygiene, behavior-cognitive therapy and sleep

  5. Enzymatic properties and localization of motopsin (PRSS12), a protease whose absence causes mental retardation. (United States)

    Mitsui, Shinichi; Yamaguchi, Nozomi; Osako, Yoji; Yuri, Kazunari


    Motopsin (PRSS12) is a mosaic protease expressed in the central nervous system. Truncation of the human motopsin gene causes nonsyndromic mental retardation. Understanding the enzymatic properties and localization of motopsin protein in the central nervous system will help identify the molecular mechanism by which the loss of motopsin function causes mental retardation. Recombinant motopsin showed amidolytic activity against the synthetic substrate benzyloxycarbonyl-l-phenylalanyl-l-arginine 4-methyl-coumaryl-7-amide. Motopsin activated the single-chain tissue plasminogen activator precursor and exhibited gelatinolytic activity. This enzymatic activity was inhibited by typical serine protease inhibitors such as aprotinin, leupeptin, and (4-amidinophenyl) methanesulfonyl fluoride. Immunocytochemistry using anti-motopsin IgG revealed that both human and mouse motopsin proteins were distributed in discrete puncta along the dendrites and soma as well as axons in cultured hippocampal neurons. In the limbic system, including the cingulate and hippocampal pyramidal neurons and piriform cortex, high level of motopsin protein was expressed at postnatal day 10, but a very low level at 10-week-old mice. Motopsin and tissue plasminogen activator were co-expressed in the cingulate pyramidal neurons at postnatal day 10 and were distributed along dendrites of cultured pyramidal neurons. In cranial nuclei, a moderate level of motopsin protein was detected independently on the developmental stage. Our results suggest that motopsin has multiple functions, such as axon outgrowth, arranging perineuronal environment, and maintaining neuronal plasticity, partly in coordination with other proteases including tissue plasminogen activator.

  6. Indispensable Role of Proteases in Plant Innate Immunity. (United States)

    Balakireva, Anastasia V; Zamyatnin, Andrey A


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

  7. Gut proteases target Yersinia invasin in vivo

    Directory of Open Access Journals (Sweden)

    Freund Sandra


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

  8. Structures of a bi-functional Kunitz-type STI family inhibitor of serine and aspartic proteases: Could the aspartic protease inhibition have evolved from a canonical serine protease-binding loop? (United States)

    Guerra, Yasel; Valiente, Pedro A; Pons, Tirso; Berry, Colin; Rudiño-Piñera, Enrique


    Bi-functional inhibitors from the Kunitz-type soybean trypsin inhibitor (STI) family are glycosylated proteins able to inhibit serine and aspartic proteases. Here we report six crystal structures of the wild-type and a non-glycosylated mutant of the bifunctional inhibitor E3Ad obtained at different pH values and space groups. The crystal structures show that E3Ad adopts the typical β-trefoil fold of the STI family exhibiting some conformational changes due to pH variations and crystal packing. Despite the high sequence identity with a recently reported potato cathepsin D inhibitor (PDI), three-dimensional structures obtained in this work show a significant conformational change in the protease-binding loop proposed for aspartic protease inhibition. The E3Ad binding loop for serine protease inhibition is also proposed, based on structural similarity with a novel non-canonical conformation described for the double-headed inhibitor API-A from the Kunitz-type STI family. In addition, structural and sequence analyses suggest that bifunctional inhibitors of serine and aspartic proteases from the Kunitz-type STI family are more similar to double-headed inhibitor API-A than other inhibitors with a canonical protease-binding loop. Copyright © 2016. Published by Elsevier Inc.

  9. HIV-1 protease-substrate coevolution in nelfinavir resistance. (United States)

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


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

  10. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

    International Nuclear Information System (INIS)

    Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F.


    The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.

  11. A novel cell-based assay to measure activity of Venezuelan equine encephalitis virus nsP2 protease

    Energy Technology Data Exchange (ETDEWEB)

    Campos-Gomez, Javier; Ahmad, Fahim; Rodriguez, Efrain; Saeed, Mohammad F., E-mail:


    The encephalitic alphaviruses encode nsP2 protease (nsP2pro), which because of its vital role in virus replication, represents an attractive target for therapeutic intervention. To facilitate the discovery of nsP2 inhibitors we have developed a novel assay for quantitative measurement of nsP2pro activity in a cell-based format. The assay is based on a substrate fusion protein consisting of eGFP and Gaussia luciferase (Gluc) linked together by a small peptide containing a VEEV nsp2pro cleavage sequence. The expression of the substrate protein in cells along with recombinant nsP2pro results in cleavage of the substrate protein resulting in extracellular release of free Gluc. The Gluc activity in supernatants corresponds to intracellular nsP2pro-mediated substrate cleavage; thus, providing a simple and convenient way to quantify nsP2pro activity. Here, we demonstrate potential utility of the assay in identification of nsP2pro inhibitors, as well as in investigations related to molecular characterization of nsP2pro. - Highlights: • A novel cell-based assay to measure VEEV nsP2 protease activity was developed. • Assay utility was demonstrated for antiviral screening. • .The assay also proved to be useful in basic mechanistic studies of nsP2 protease.


    Directory of Open Access Journals (Sweden)

    R. Satheeskumar


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

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

    International Nuclear Information System (INIS)

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


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

  14. Isolation, cDNA Cloning, and Structure-based Functional Characterization of Oryctin, a Hemolymph Protein from the Coconut Rhinoceros Beetle, Oryctes rhinoceros, as a Novel Serine Protease Inhibitor* (United States)

    Horita, Shoichiro; Ishibashi, Jun; Nagata, Koji; Miyakawa, Takuya; Yamakawa, Minoru; Tanokura, Masaru


    We isolated oryctin, a 66-residue peptide, from the hemolymph of the coconut rhinoceros beetle Oryctes rhinoceros and cloned its cDNA. Oryctin is dissimilar to any other known peptides in amino acid sequence, and its function has been unknown. To reveal that function, we determined the solution structure of recombinant 13C,15N-labeled oryctin by heteronuclear NMR spectroscopy. Oryctin exhibits a fold similar to that of Kazal-type serine protease inhibitors but has a unique additional C-terminal α-helix. We performed protease inhibition assays of oryctin against several bacterial and eukaryotic proteases. Oryctin does inhibit the following serine proteases: α-chymotrypsin, endopeptidase K, subtilisin Carlsberg, and leukocyte elastase, with Ki values of 3.9 × 10−10 m, 6.2 × 10−10 m, 1.4 × 10−9 m, and 1.2 × 10−8 m, respectively. Although the target molecule of oryctin in the beetle hemolymph remains obscure, our results showed that oryctin is a novel single domain Kazal-type inhibitor and could play a key role in protecting against bacterial infections. PMID:20630859

  15. Supermarket Proteases. (United States)

    Hagar, William G.; Bullerwell, Lornie D.


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

  16. Contemporary protease inhibitors and cardiovascular risk

    DEFF Research Database (Denmark)

    Lundgren, Jens; Mocroft, Amanda; Ryom, Lene


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

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

    KAUST Repository

    Li, Zhao


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

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

    KAUST Repository

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


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

  19. Antiviral Potential of a Novel Compound CW-33 against Enterovirus A71 via Inhibition of Viral 2A Protease

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    Ching-Ying Wang


    Full Text Available Enterovirus A71 (EV-A71 in the Picornaviridae family causes hand-foot-and-mouth disease, aseptic meningitis, severe central nervous system disease, even death. EV-A71 2A protease cleaves Type I interferon (IFN-α/β receptor 1 (IFNAR1 to block IFN-induced Jak/STAT signaling. This study investigated anti-EV-A7l activity and synergistic mechanism(s of a novel furoquinoline alkaloid compound CW-33 alone and in combination with IFN-β Anti-EV-A71 activities of CW-33 alone and in combination with IFN-β were evaluated by inhibitory assays of virus-induced apoptosis, plaque formation, and virus yield. CW-33 showed antiviral activities with an IC50 of near 200 µM in EV-A71 plaque reduction and virus yield inhibition assays. While, anti-EV-A71 activities of CW-33 combined with 100 U/mL IFN-β exhibited a synergistic potency with an IC50 of approximate 1 µM in plaque reduction and virus yield inhibition assays. Molecular docking revealed CW-33 binding to EV-A71 2A protease active sites, correlating with an inhibitory effect of CW33 on in vitro enzymatic activity of recombinant 2A protease IC50 = 53.1 µM. Western blotting demonstrated CW-33 specifically inhibiting 2A protease-mediated cleavage of IFNAR1. CW-33 also recovered Type I IFN-induced Tyk2 and STAT1 phosphorylation as well as 2',5'-OAS upregulation in EV-A71 infected cells. The results demonstrated CW-33 inhibiting viral 2A protease activity to reduce Type I IFN antagonism of EV-A71. Therefore, CW-33 combined with a low-dose of Type I IFN could be applied in developing alternative approaches to treat EV-A71 infection.

  20. Age-specific incidence rates for dementia and Alzheimer disease in NIA-LOAD/NCRAD and EFIGA families: National Institute on Aging Genetics Initiative for Late-Onset Alzheimer Disease/National Cell Repository for Alzheimer Disease (NIA-LOAD/NCRAD) and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA). (United States)

    Vardarajan, Badri N; Faber, Kelley M; Bird, Thomas D; Bennett, David A; Rosenberg, Roger; Boeve, Bradley F; Graff-Radford, Neill R; Goate, Alison M; Farlow, Martin; Sweet, Robert A; Lantigua, Rafael; Medrano, Martin Z; Ottman, Ruth; Schaid, Daniel J; Foroud, Tatiana M; Mayeux, Richard


    Late-onset Alzheimer disease (LOAD), defined as onset of symptoms after age 65 years, is the most common form of dementia. Few reports investigate incidence rates in large family-based studies in which the participants were selected for family history of LOAD. To determine the incidence rates of dementia and LOAD in unaffected members in the National Institute on Aging Genetics Initiative for Late-Onset Alzheimer Disease/National Cell Repository for Alzheimer Disease (NIA-LOAD/NCRAD) and Estudio Familiar de Influencia Genetica en Alzheimer (EFIGA) family studies. Families with 2 or more affected siblings who had a clinical or pathological diagnosis of LOAD were recruited as a part of the NIA-LOAD/NCRAD Family Study. A cohort of Caribbean Hispanics with familial LOAD was recruited in a different study at the Taub Institute for Research on Alzheimer's Disease and the Aging Brain in New York and from clinics in the Dominican Republic as part of the EFIGA study. Age-specific incidence rates of LOAD were estimated in the unaffected family members in the NIA-LOAD/NCRAD and EFIGA data sets. We restricted analyses to families with follow-up and complete phenotype information, including 396 NIA-LOAD/NCRAD and 242 EFIGA families. Among the 943 at-risk family members in the NIA-LOAD/NCRAD families, 126 (13.4%) developed dementia, of whom 109 (86.5%) met criteria for LOAD. Among 683 at-risk family members in the EFIGA families, 174 (25.5%) developed dementia during the study period, of whom 145 (83.3%) had LOAD. The annual incidence rates of dementia and LOAD in the NIA-LOAD/NCRAD families per person-year were 0.03 and 0.03, respectively, in participants aged 65 to 74 years; 0.07 and 0.06, respectively, in those aged 75 to 84 years; and 0.08 and 0.07, respectively, in those 85 years or older. Incidence rates in the EFIGA families were slightly higher, at 0.03 and 0.02, 0.06 and 0.05, 0.10 and 0.08, and 0.10 and 0.07, respectively, in the same age groups. Contrasting these

  1. Detergent-compatible proteases: microbial production, properties, and stain removal analysis. (United States)

    Niyonzima, Francois Niyongabo; More, Sunil


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


    Directory of Open Access Journals (Sweden)

    Roberval Rodrigues da Costa


    Full Text Available

    This investigation was carried on in six counties of milk region around Goiânia city, state of Goiás. Seven hundred and one (701 cows in lactation were examined, from which 87 (12.41% showed c1inica1 and subclinical mastitis. The tests CMT/ Whiteside were dome on 2,717 samples of milk and from those 393 (41.46% were positive. The rnicroorganisms isolated from 701 lactocultures were: Staphilococcus aureus 172 (67.70%; Streptococcus spp. 38 (l4.96%; Corynebacterium spp. 42 (16.53%; Staphylococcu.s epidermides 38 (14.96%; Pseudomonas aeruginosa 21 (8.26%; Eschericheir coli 18 (7.08%; Serratia marceceus 12 (4.72%; Klebsiella spp 5 (1.96%; Proteus vulgaris 4 (1.57%; Candida spp 5 (1.96%; Candida spp 5 (1.96% and in 23 (9.05% there was no growing.

    Esta pesquisa foi realizada em seis municípios da bacia leiteira de Goiânia tendo sido examinadas 701 vacas lactantes, das quais 87 (12,41% apresentaram mastite clínica e subclínica. Os testes CMT/Whiteside foram realizados em 2.717 amostras de leite, sendo que 393 (14,46% resultaram positivas. Os microrganismos isolados das 701 lactoculturas foram: Staphilococcus aureus 172 (67,70%; Streptococcus spp. 38 (l4,96%; Corynebacterium spp. 42 (16 ,53%; Staphylococcu.s epidermides 38 (14 ,96%; Pseudomonas aeruginosa 21 (8,26%; Eschericheir coli 18 (7,08%; Serratia marceceus 12 (4,72%; Klebsiella spp 5 (1,96%; Proteus vulgaris 4 (1,57%; Candida spp 5 (1,96%; Candida spp 5 (1,96% e em 23 (9,05% não houve crescimento.

  3. The Effect of Exogenous Protease in Broiler Diets on the Apparent Ileal Digestibility of Amino Acids and on Protease Activity in Jejunum

    Directory of Open Access Journals (Sweden)

    Vojtěch Rada


    Full Text Available The objective of this study was to evaluate the effect of a mono-component commercial serine protease supplement in broiler diets on apparent ileal amino acid digestibility and protease activity. A total of 150 male (28 d old ROSS 308 were randomly placed into 30 battery pens and divided into 5 treatment groups with 6 replicates each. The experiment was performed for 7 days. Five dietary treatments were used: 2 standard protein diets without (SP and with protease (SP + P formulated 20.7 % CP, 2 lower-protein diets (19.9 % CP without (LP and with protease (LP + P and one lower‑protein diet with protease and with doubled rapeseed meal (RSM content (SP-RSM + P compared with the other treatments. Lower-protein diets were formulated with a 4 % decrease in the relative CP value compared with the standard protein diet. Enzyme protease was added to the diets at a concentration of 200 ppm (15,000 PROT units per kg. The diets contained 0.3 % Cr2O3 to facilitate the estimation of apparent AA digestibility and overall apparent ileal crude protein digestibility. Mono-component protease had no effect on apparent ileal AA digestibility or jejunum protease activity if diets contained the same level of RSM. The supplement of exogenous protease did not affect (P > 0.05 the apparent ileal AA digestibility coefficients if a higher RSM level was used. The CP level influenced (P < 0.05 only the coefficients of the apparent ileal AA digestibility of Pro and Arg. The RSM level (P < 0.01 had significant effects on protease activity in the jejunum.

  4. Pulmonary deposition and disappearance of aerosolised secretory leucocyte protease inhibitor. (United States)

    Stolk, J.; Camps, J.; Feitsma, H. I.; Hermans, J.; Dijkman, J. H.; Pauwels, E. K.


    BACKGROUND--The neutrophil elastase inhibitor, secretory leucocyte protease inhibitor (SLPI), is a potential therapeutic tool in inflammatory lung diseases such as cystic fibrosis and pulmonary emphysema. The distribution and disappearance in the lung of aerosolised recombinant SLPI (rSLPI) was investigated in healthy humans and in patients with cystic fibrosis or alpha 1-antitrypsin-associated emphysema. METHODS--To distinguish aerosolised rSLPI from endogenous SLPI the recombinant inhibitor was radiolabelled with 99m-technetium (99mTc) pertechnetate. Distribution and disappearance of aerosolised 99mTc-rSLPI in the lungs were studied by gamma radiation imaging. RESULTS--The deposition of 99mTc-rSLPI in normal volunteers was homogeneous in all lung lobes, while in patients with cystic fibrosis or emphysema only well ventilated areas showed deposition of the aerosol. The disappearance rate of 99mTc-rSLPI was biexponential. The half life of the rapid phase was 0.2-2.8 hours, while that of the slow phase was more than 24 hours. CONCLUSIONS--Future aerosol therapy with rSLPI will be most beneficial for well ventilated lung tissue that needs protection against neutrophil derived elastase. It may be more difficult to neutralise the burden of elastase in poorly ventilated, highly inflamed areas as are seen in cystic fibrosis. Images PMID:7638807

  5. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    International Nuclear Information System (INIS)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui; Leppla, Stephen H.


    Highlights: ► Non-infectious and protease-deficient Bacillus anthracis protein expression system. ► Successful expression and purification of a tumor-targeted fusion protein drug. ► Very low endotoxin contamination of purified protein. ► Efficient protein secretion simplifies purification. ► Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGFα). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGFα). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  6. O desmatamento na Amazônia e a importância das áreas protegidas

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    Leandro Valle Ferreira


    Full Text Available O MODELO da ocupação demográfica da Amazônia legalnos últimos cinqueta anos tem levado a níveis significativos de desmatamento, resultante de múltiplos fatores, tais como a abertura de estradas pioneiras, o crescimento das cidades, a ampliação de pecuária extensiva, a acelerada exploração madeireira e a crescente agricultura intensiva de monoculturas. A área cumulativa desmatada na Amazônia legal brasileira chegou a cerca de 653.000 km² em 2003, correspondendo a 16,3% da região. Este estudo visou a determinar o desmatamento dentro e fora dos atuais Unidades de Conservação (UC e Terras Indígenas (TI na Amazônia legal, nos estados de Mato Grosso, Rondônia e Pará, que, juntos, corresponderam por mais de 90% do desmatamento observado entre 2001 e 2003. Os resultados mostraram que o desmatamento foi cerca de dez a vinte vezes menor dentro das Unidades de Conservação e Terras Indígenas do que em áreas contíguas fora delas. Isto demonstra a importância dessas áreas protegidas para diminuir o processo do desmatamento nos três estados. Isto refuta a hipótese generalizada de que estas áreas não cumprissem a sua função principal na conservação e uso racional dos recursos na Amazônia legal.DEMOGRAPHIC occupation of the Brazilian Legal Amazon within the past 50 years has lead to significant levels of deforestation as the result of several factors such as new road construction, urban growth, expansion of extensive cattle ranching, increasing timber extraction, and rising intensive agriculture of monocultures. The cumulative deforested area in Brazil's Legal Amazonia was approximately 653,000 km² in 2003, correspondingto 16,3% of the region. This study sought to assess deforestation within or near to protected areas (conservation units and indigenous reserves in Legal Amazonia in the states of Mato Grosso, Rondônia, and Pará that jointly were responsible for more than 90% of regional deforestation from 2001 to 2003

  7. SmCL3, a gastrodermal cysteine protease of the human blood fluke Schistosoma mansoni.

    Directory of Open Access Journals (Sweden)

    Jan Dvorák


    Full Text Available Blood flukes of the genus Schistosoma are platyhelminth parasites that infect 200 million people worldwide. Digestion of nutrients from the host bloodstream is essential for parasite development and reproduction. A network of proteolytic enzymes (proteases facilitates hydrolysis of host hemoglobin and serum proteins.We identified a new cathepsin L termed SmCL3 using PCR strategies based on S. mansoni EST sequence data. An ortholog is present in Schistosoma japonicum. SmCL3 was heterologously expressed as an active enzyme in the yeast, Pichia pastoris. Recombinant SmCL3 has a broad pH activity range against peptidyl substrates and is inhibited by Clan CA protease inhibitors. Consistent with a function in degrading host proteins, SmCL3 hydrolyzes serum albumin and hemoglobin, is localized to the adult gastrodermis, and is expressed mainly in those life stages infecting the mammalian host. The predominant form of SmCL3 in the parasite exists as a zymogen, which is unusual for proteases. This zymogen includes an unusually long prodomain with alpha helical secondary structure motifs. The striking specificity of SmCL3 for amino acids with large aromatic side chains (Trp and Tyr at the P2 substrate position, as determined with positional scanning-synthetic combinatorial library, is consistent with a molecular model that shows a large and deep S2 pocket. A sequence similarity network (SSN view clusters SmCL3 and other cathepsins L in accordance with previous large-scale phylogenetic analyses that identify six super kingdoms.SmCL3 is a gut-associated cathepsin L that may contribute to the network of proteases involved in degrading host blood proteins as nutrients. Furthermore, this enzyme exhibits some unusual sequence and biophysical features that may result in additional functions. The visualization of network inter-relationships among cathepsins L suggests that these enzymes are suitable 'marker sequences' for inclusion in future phylogenetic analyses.

  8. Jornada sobre Riscos Penals de la Banca en Línia

    Directory of Open Access Journals (Sweden)

    Rosa Fernández


    Full Text Available

    Ressenya de la jornada organitzada el desembre del 2005 sobre els nous riscos a què la banca en línia i el comerç electrònic estan sotmesos a causa de l'augment d'atacs de tipus pesca electrònica o phishing i altres defraudacions semblants.

  9. Perubahan Dan Keberlanjutan Tari Balanse Madam Di Lingkungan Masyarakat Nias Padang

    Directory of Open Access Journals (Sweden)

    Novina Yeni Fatrina


    Full Text Available Tulisan berjudul “Perubahan dan Keberlanjutan Tari Balanse Madam di Lingkungan Masyarakat Nias Padang“ bertujuan untuk mengungkapkan perubahan  tari Balanse Madam serta menganalisis keberlanjutannya. Tari Balanse Madam merupakan tarian kelompok yang terdiri dari pasangan penari laki-laki dan perempuan yang sudah menikah. Penampilannya diiringi secara live dengan alat musik barat seperti biola, gitar, tamburin, akordeon, dan set drum. Namun setelah banyak para pelaku seninya yang meninggal dunia, sehingga tari Balanse Madam mengalami perubahan. Perubahan terjadi pada penarinya, tidak harus orang yang sudah berumahtangga lagi, sedangkan musik iringannya no use life music. Analisa perubahan tari Balanse Madam diamati dari  tahun 1995 sampai tahun 2017.   Metode penelitian deskripsi kualitatif dengan pendekatan interaksi dan interpretasi analisis digunakan untuk mengungkapkan permasalahan tersebut. Pelacakan dilakukan dengan melihat dan menganalisa perubahan tari Balanse Madam pada perkembangan elemen pembentuk komposisi tarinya dari tahun 1995 sampai tahun 2017. Penelitian perubahan dan keberlanjutan tari Balanse Madam di lingkungan masyarakat Nias Padang dianalisis dengan menggunakan teori ketahanan budaya yang dikemukakan oleh Edi Sedyawati dan teori elemen pembentuk komposisi yang digunakan oleh R.M. Soedarsono. Hasil penelitian menunjukan bahwa seiring dengan berjalannya waktu, berbagai hal mempengaruhi kondisi tari Balanse Madam, sehingga terjadi perubahan pada beberapa elemen pembentuk tari Balanse Madam. Adapun perubahan tersebut adalah (1 penari terdiri orang-orang yang masih remaja; (2 musik iringan mengalami perubahan irama dan terkadang diiringi musik rekaman. Pemakaian penari remaja dan musik rekaman merupakan salah satu bentuk keberlanjutan tari Balanse Madam. Inilah yang membuat tari Balanse Madam masih tetap bertahan dalam kehidupan masyarakat Nias Padang. The article entitled "The Change and Sustainability of Balanse Madam

  10. Geotecnologias aplicadas ao estudo de precipita??o e desmatamento na por??o Sul da Amaz?nia


    Seixas, Ian Marins


    Na Amaz?nia, os impactos das diversas atividades (extra??o de madeira, minera??o e agropecu?ria) sobre a floresta geram a destrui??o da biodiversidade, a degrada??o dos solos e poss?veis altera??es clim?ticas. A floresta possui uma intensa intera??o com a atmosfera ao fornecer uma grande quantidade de calor e vapor d ?gua contribuindo para a umidade local. Considerando o atual cen?rio de desmatamento no sul da Amaz?nia (Arco do Desmatamento), tem-se como hip?tese que a transforma??o do uso da...

  11. A new method for the characterization of strain-specific conformational stability of protease-sensitive and protease-resistant PrPSc.

    Directory of Open Access Journals (Sweden)

    Laura Pirisinu

    Full Text Available Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrP(Sc, a disease-associated isoform of the host-encoded cellular protein (PrP(C. Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc. However, PrP(Sc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrP(Sc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C and PrP(Sc by means of differential centrifugation. The conformational solubility and stability assay (CSSA was then developed by measuring PrP(Sc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl](1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl](1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M, followed by sheep scrapie (2.2 M and by MM2 sCJD (1.6 M. In order to test the ability of CSSA to characterise protease-sensitive PrP(Sc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrP(Sc conformational stabilities of protease-resistant and protease-sensitive PrP(Sc and that it is a valuable tool

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

    International Nuclear Information System (INIS)

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


    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.

  13. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

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    Suk Ho Choi


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

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

    International Nuclear Information System (INIS)

    Adachi, Motoyasu; Kuroki, Ryota


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

  15. O "abrasileiramento" das associações esportivas de Teutônia/Estrela no Rio Grande do Sul The "abrasileiramento" of the sports associations from Teutônia/Estrela in Rio Grande do Sul

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    Cecília Elisa Kilpp


    Full Text Available As associações esportivas teuto-brasileiras de Teutônia/Estrela no Estado do Rio Grande do Sul sofreram o processo de abrasileiramento no período da Primeira Guerra Mundial (1914-1918 e da Segunda Grande Guerra (1939-1945. Este artigo objetivou identificar os conflitos de identidades culturais nas associações esportivas de Teutônia/Estrela desencadeados pelas ações nacionalizadoras conduzidas pelos interventores do Estado Rio Grande do Sul no período das grandes guerras mundiais. As fontes históricas consultadas revelaram que a prática de esportes tradicionais nas associações teuto-brasileiras enfraqueceu, como no caso do bolão, ao mesmo tempo que outros esportes foram incorporados, principalmente o futebol. A adesão a esta nova prática esportiva significa um movimento na direção do processo de integração às ações nacionalizadoras.The German-Brazilian sport associations of Teutônia/Estrela in the State of Rio Grande do Sul suffered the process of abrasileiramento in the period of the World War I (1914-1918 and of the World War II (1939-1945. This article aimed to identify the conflicts of cultural identities in the sport associations of Teutônia/Estrela developed by the nationalization actions leaded by the intervenors of the Rio Grande do Sul State in the period of the great world wars. Historical sources revealed that the practice of traditional sports in the German-Brazilian sport associations weakened, asthe bolão, while other sports were incorporated, mainly the soccer. The adhesion to this new sport means a movement in the direction of the process of integration in the nationalization actions.

  16. Texto, textualidades e jornalismo: a legitimação de práticas culturais nos editoriais do Diário Folha de Rondônia

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    Colferai, Sandro Adalberto


    Full Text Available Este artigo aborda a tomada de sentidos preferenciais em textos jornalísticos produzidos e em circulação na região amazônica. O recorte é feito a partir de textos de editoriais publicados pelo jornal diário Folha de Rondônia, editado no interior de Rondônia. A análise dos textos é feita a partir da perspectiva dos Estudos Culturais, como apresentada por Stuart Hall, Richard Johnson e Nick Couldry. São tomados os conceitos de Cultura, Identidade Cultural e Texto, além de se recuperar parte da história social da Amazônia e, em especial, de Rondônia, esta imersa no choque entre diferentes práticas simbólicas de populações tradicionais da Amazônia e colonizadores que passaram a ocupá-la a partir da década de 1970

  17. Construction of dengue virus protease expression plasmid and in vitro protease assay for screening antiviral inhibitors. (United States)

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan


    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.

  18. Gene and process level modulation to overcome the bottlenecks of recombinant proteins expression in Pichia pastoris. (United States)

    Prabhu, Ashish A; Boro, Bibari; Bharali, Biju; Chakraborty, Shuchishloka; Dasu, V Venkata


    Process development involving system metabolic engineering and bioprocess engineering has become one of the major thrust for the development of therapeutic proteins or enzymes. Pichia pastoris has emerged as a prominent host for the production of therapeutic protein or enzymes. Despite of producing high protein titers, various cellular and process level bottlenecks hinders the expression of recombinant proteins in P. pastoris. In the present review, we have summarized the recent developments in the expression of foreign proteins in P. pastoris. Further, we have discussed various cellular engineering strategies which include codon optimization, pathway engineering, signal peptide processing, development of protease deficient strain and glyco-engineered strains for the high yield protein secretion of recombinant protein. Bioprocess development of recombinant proteins in large scale bioreactor including medium optimization, optimum feeding strategy and co-substrate feeding in fed batch as well as continuous cultivation have been described. The recent advances in system and synthetic biology studies including metabolic flux analysis in understanding the phenotypic characteristics of recombinant Pichia and genome editing with CRISPR-CAS system have also been summarized. Copyright© Bentham Science Publishers; For any queries, please email at

  19. Multifunctional Mitochondrial AAA Proteases. (United States)

    Glynn, Steven E


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

  20. Identification and characterization of human polyserase-3, a novel protein with tandem serine-protease domains in the same polypeptide chain

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    Garabaya Cecilia


    Full Text Available Abstract Background We have previously described the identification and characterization of polyserase-1 and polyserase-2, two human serine proteases containing three different catalytic domains within the same polypeptide chain. Polyserase-1 shows a complex organization and it is synthesized as a membrane-bound protein which can generate three independent serine protease domains as a consequence of post-translational processing events. The two first domains are enzymatically active. By contrast, polyserase-2 is an extracellular glycosylated protein whose three protease domains remain embedded in the same chain, and only the first domain possesses catalytic activity. Results Following our interest in the study of the human degradome, we have cloned a human liver cDNA encoding polyserase-3, a new protease with tandem serine protease domains in the same polypeptide chain. Comparative analysis of polyserase-3 with the two human polyserases described to date, revealed that this novel polyprotein is more closely related to polyserase-2 than to polyserase-1. Thus, polyserase-3 is a secreted protein such as polyserase-2, but lacks additional domains like the type II transmembrane motif and the low-density lipoprotein receptor module present in the membrane-anchored polyserase-1. Moreover, analysis of post-translational mechanisms operating in polyserase-3 maturation showed that its two protease domains remain as integral parts of the same polypeptide chain. This situation is similar to that observed in polyserase-2, but distinct from polyserase-1 whose protease domains are proteolytically released from the original chain to generate independent units. Immunolocalization studies indicated that polyserase-3 is secreted as a non-glycosylated protein, thus being also distinct from polyserase-2, which is a heavily glycosylated protein. Enzymatic assays indicated that recombinant polyserase-3 degrades the α-chain of fibrinogen as well as pro

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

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    Lilburn Timothy G


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

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


    Yusriah, Yusriah; Kuswytasari, Nengah Dwianita


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

  3. Mini review: Recombinant production of tailored bio-pharmaceuticals in different Bacillus strains and future perspectives. (United States)

    Lakowitz, Antonia; Godard, Thibault; Biedendieck, Rebekka; Krull, Rainer


    Bio-pharmaceuticals like antibodies, hormones and growth factors represent about one-fifth of commercial pharmaceuticals. Host candidates of growing interest for recombinant production of these proteins are strains of the genus Bacillus, long being established for biotechnological production of homologous and heterologous proteins. Bacillus strains benefit from development of efficient expression systems in the last decades and emerge as major industrial workhorses for recombinant proteins due to easy cultivation, non-pathogenicity and their ability to secrete recombinant proteins directly into extracellular medium allowing cost-effective downstream processing. Their broad product portfolio of pharmaceutically relevant recombinant proteins described in research include antibody fragments, growth factors, interferons and interleukins, insulin, penicillin G acylase, streptavidin and different kinases produced in various cultivation systems like microtiter plates, shake flasks and bioreactor systems in batch, fed-batch and continuous mode. To further improve production and secretion performance of Bacillus, bottlenecks and limiting factors concerning proteases, chaperones, secretion machinery or feedback mechanisms can be identified on different cell levels from genomics and transcriptomics via proteomics to metabolomics and fluxomics. For systematical identification of recurring patterns characteristic of given regulatory systems and key genetic targets, systems biology and omics-technology provide suitable and promising approaches, pushing Bacillus further towards industrial application for recombinant pharmaceutical protein production. Copyright © 2017. Published by Elsevier B.V.

  4. Functional protease profiling for diagnosis of malignant disease. (United States)

    Findeisen, Peter; Neumaier, Michael


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


    Directory of Open Access Journals (Sweden)

    Carolina Ferreira da Costa STREGLIO


    Full Text Available Nas últimas décadas, o crescente movimento de proteção ambiental vem interferindo na configuração das cidades, conjuntura em que se destaca a criação de parques urbanos. As funções ecológicas e sociais atribuídas a essas áreas verdes as tornam objetos de estudo de grande relevância para compreender a dinâmica urbana atual, contexto em que se insere o município de Goiânia. Considerando esses atributos, bem como o caráter distintivo que os parques estabelecem na paisagem urbana, o que permite relacioná-los também ao turismo, este artigo tem como objetivo analisar o desempenho dos parques urbanos na dinâmica de Goiânia, para, com base nessa análise, verificar o seu potencial turístico.

  6. Identification of cysteine proteases and screening of cysteine protease inhibitors in biological samples by a two-dimensional gel system of zymography and reverse zymography. (United States)

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


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

  7. Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients. (United States)

    Buhner, Sabine; Hahne, Hannes; Hartwig, Kerstin; Li, Qin; Vignali, Sheila; Ostertag, Daniela; Meng, Chen; Hörmannsperger, Gabriele; Braak, Breg; Pehl, Christian; Frieling, Thomas; Barbara, Giovanni; De Giorgio, Roberto; Demir, Ihsan Ekin; Ceyhan, Güralp Onur; Zeller, Florian; Boeckxstaens, Guy; Haller, Dirk; Kuster, Bernhard; Schemann, Michael


    The causes of gastrointestinal complaints in irritable bowel syndrome (IBS) remain poorly understood. Altered nerve function has emerged as an important pathogenic factor as IBS mucosal biopsy supernatants consistently activate enteric and sensory neurons. We investigated the neurally active molecular components of such supernatants from patients with IBS and quiescent ulcerative colitis (UC). Effects of supernatants from 7 healthy controls (HC), 20 IBS and 12 UC patients on human and guinea pig submucous neurons were studied with neuroimaging techniques. We identify differentially expressed proteins with proteome analysis. Nerve activation by IBS supernatants was prevented by the protease activated receptor 1 (PAR1) antagonist SCHE79797. UC supernatants also activated enteric neurons through protease dependent mechanisms but without PAR1 involvement. Proteome analysis of the supernatants identified 204 proteins, among them 17 proteases as differentially expressed between IBS, UC and HC. Of those the four proteases elastase 3a, chymotrypsin C, proteasome subunit type beta-2 and an unspecified isoform of complement C3 were significantly more abundant in IBS compared to HC and UC supernatants. Of eight proteases, which were upregulated in IBS, the combination of elastase 3a, cathepsin L and proteasome alpha subunit-4 showed the highest prediction accuracy of 98% to discriminate between IBS and HC groups. Elastase synergistically potentiated the effects of histamine and serotonin-the two other main neuroactive substances in the IBS supernatants. A serine protease inhibitor isolated from the probiotic Bifidobacterium longum NCC2705 (SERPINBL), known to inhibit elastase-like proteases, prevented nerve activation by IBS supernatants. Proteases in IBS and UC supernatants were responsible for nerve activation. Our data demonstrate that proteases, particularly those signalling through neuronal PAR1, are biomarker candidates for IBS, and protease profiling may be used to

  8. Serine protease inhibitors of parasitic helminths. (United States)

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


    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.

  9. Analysis of Milk from Mothers Who Delivered Prematurely Reveals Few Changes in Proteases and Protease Inhibitors across Gestational Age at Birth and Infant Postnatal Age. (United States)

    Demers-Mathieu, Veronique; Nielsen, Søren Drud; Underwood, Mark A; Borghese, Robyn; Dallas, David C


    Background: Peptidomics research has demonstrated that protease activity is higher in breast milk from preterm-delivering mothers than from term-delivering mothers. However, to our knowledge, the effect of the degree of prematurity and postnatal age on proteases and protease inhibitors in human milk remains unknown. Objective: We aimed to determine the change of proteases and protease inhibitors in milk from mothers who delivered prematurely across gestational age (GA) and postnatal age. Methods: Milk samples were collected from 18 mothers aged 26-40 y who delivered preterm infants and who lacked mastitis. For analysis, samples were separated into 2 groups: 9 from early GA (EGA) (24-26 wk GA)-delivering mothers and 9 from late GA (LGA) (27-32 wk GA)-delivering mothers. Within the 9 samples in each group, the collection time ranged from postnatal days 2 to 47. The activity and predicted activity of proteases in preterm milk were determined with the use of fluorometric and spectrophotometric assays and peptidomics, respectively. Protease and protease inhibitor concentrations were determined with the use of ELISA. Linear mixed models were applied to compare enzymes across GA and postnatal age. Results: Carboxypeptidase B2, kallikrein, plasmin, elastase, thrombin, and cytosol aminopeptidase were present and active in the milk of preterm-delivering mothers. Most milk protease and antiprotease concentrations did not change with GA or postnatal age. However, the concentration and activity of kallikrein, the most abundant and active protease in preterm milk, increased by 25.4 ng · mL -1 · d -1 and 0.454 μg · mL -1 · d -1 postnatally, respectively, in EGA milk samples while remaining stable in LGA milk samples. Conclusions: This research demonstrates that proteases are active in human milk and begin to degrade milk protein within the mammary gland before consumption by infants. Proteases and protease inhibitors in milk from mothers of premature infants mostly did not

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


    Naiola, Elidar; Widhyastuti, Nunuk


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

  11. Influência da salinidade da água no crescimento de seis genótipos de helicônias

    Directory of Open Access Journals (Sweden)

    Helder M. M. Barros


    Full Text Available No Nordeste brasileiro a qualidade das águas utilizadas em irrigação é muito variável tanto em termos geográficos como ao longo do ano, principalmente em pequenos açudes. Com a utilização de águas com altos níveis de salinidade aliada às condições climáticas, comumente encontradas no final do período de estiagem, há riscos de salinização dos solos irrigados com prejuízos, também, para as culturas. Para helicônias, não existem quaisquer indicativos relacionados ao efeito da salinidade da água no desenvolvimento. Propôs-se, neste trabalho, avaliar o crescimento na altura de planta mediante altura, número de folhas, teor de água na planta e fitomassa seca de seis genótipos de helicônias irrigados com águas salinas. O experimento foi conduzido em casa de vegetação pertencente à UFCG/Campina Grande, PB, compondo um esquema fatorial 6 x 6 em delineamento experimental de blocos ao acaso, com três repetições, cada um com dois vasos. O crescimento das helicônias foi afetado significativamente pelos níveis de salinidade. A H. latispatha cv. Red-Yellow Gyro e a H. psittacorum cv. Red Opal foram as helicônias menos afetadas pela salinidade. O teor de água na planta diminuiu com o aumento da CE da água de irrigação. Com o aumento da salinidade, ocorreu decréscimo na fitomassa seca. A H. x nickeriensis apresentou os menores índices em comparação com as demais helicônias, sendo mais sensível à salinidade.

  12. HIV protease drug resistance and its impact on inhibitor design. (United States)

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


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

  13. Pressure sound level measurements at an educational environment in Goiânia, Goiás, Brazil (United States)

    Costa, J. J. L.; do Nascimento, E. O.; de Oliveira, L. N.; Caldas, L. V. E.


    In this work, 25 points located on the ground floor of the Federal Institute of Education, Science and Technology of Goias - IFG - Campus Goiânia, were analyzed in morning periods of two Saturdays. The pressure sound levels were measured at internal and external environments during routine activities seeking to perform an environmental monitoring at this institution. The initial hypothesis was that an amusement park (Mutirama Park) was responsible for originating noise pollution in the institute, but the results showed, within the campus environment, sound pressure levels in accordance with the Municipal legislation of Goiânia for all points.

  14. Forest culicinae mosquitoes in the environs of samuel hydroeletric plant, state of Rondônia, Brazil

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    SLB Luz


    Full Text Available Data on frequency and seasonal distribution of culicinae were recorded in the forest near a recently constructed hydroelectric plant - Samuel, in the State of Rondônia, Brazilian Amazon. Collections were performed almost daily from August 1990 to July 1991, between 6 and 9 p.m., using human bait. A total of 3,769 mosquitoes was collected, representing 21 species, including seven new records for the State of Rondônia. The most frequently collected species were Aedes fulvus (25% and Ae. pennai (12.3%. The highest density for the majority of mosquito species coincided with the rainy season.

  15. 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. (United States)

    Rossano, Rocco; Larocca, Marilena; Riccio, Paolo


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

  16. Detection of protease activity in cells and animals. (United States)

    Verdoes, Martijn; Verhelst, Steven H L


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

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

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    Kanupriya Miglani Sharma


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

  18. Pathophysiological significance and therapeutic applications of snake venom protease inhibitors. (United States)

    Thakur, Rupamoni; Mukherjee, Ashis K


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

  19. Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target (United States)

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


    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.

  20. [Analysis of salivary protease spectrum in chronic periodontitis]. (United States)

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


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

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


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


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

  2. Recombinant expression and purification of a tumor-targeted toxin in Bacillus anthracis

    Energy Technology Data Exchange (ETDEWEB)

    Bachran, Christopher; Abdelazim, Suzanne; Fattah, Rasem J.; Liu, Shihui [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States); Leppla, Stephen H., E-mail: [National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892 (United States)


    Highlights: Black-Right-Pointing-Pointer Non-infectious and protease-deficient Bacillus anthracis protein expression system. Black-Right-Pointing-Pointer Successful expression and purification of a tumor-targeted fusion protein drug. Black-Right-Pointing-Pointer Very low endotoxin contamination of purified protein. Black-Right-Pointing-Pointer Efficient protein secretion simplifies purification. Black-Right-Pointing-Pointer Functional anti-tumor fusion protein purified. -- Abstract: Many recombinant therapeutic proteins are purified from Escherichia coli. While expression in E. coli is easily achieved, some disadvantages such as protein aggregation, formation of inclusion bodies, and contamination of purified proteins with the lipopolysaccharides arise. Lipopolysaccharides have to be removed to prevent inflammatory responses in patients. Use of the Gram-positive Bacillus anthracis as an expression host offers a solution to circumvent these problems. Using the multiple protease-deficient strain BH460, we expressed a fusion of the N-terminal 254 amino acids of anthrax lethal factor (LFn), the N-terminal 389 amino acids of diphtheria toxin (DT389) and human transforming growth factor alpha (TGF{alpha}). The resulting fusion protein was constitutively expressed and successfully secreted by B. anthracis into the culture supernatant. Purification was achieved by anion exchange chromatography and proteolytic cleavage removed LFn from the desired fusion protein (DT389 fused to TGF{alpha}). The fusion protein showed the intended specific cytotoxicity to epidermal growth factor receptor-expressing human head and neck cancer cells. Final analyses showed low levels of lipopolysaccharides, originating most likely from contamination during the purification process. Thus, the fusion to LFn for protein secretion and expression in B. anthracis BH460 provides an elegant tool to obtain high levels of lipopolysaccharide-free recombinant protein.

  3. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

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

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

  4. Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

    Directory of Open Access Journals (Sweden)

    Tanveer Majeed


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

  5. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.


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

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

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    Marian Dorcas Quain


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

  7. Effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus. (United States)

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


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


    Directory of Open Access Journals (Sweden)

    Saint-Clair Cordeiro da Trindade Júnior


    Full Text Available Uma fase de rápida ocupação do território foi inaugurada a partir da década de 1960 na Amazônia. A presença de empresários, especuladores e migrantes, mediados por ações intencionais do Estado, definiu uma nova forma de ocupação territorial para a região. As estratégias de ação se basearam em pelo menos três tipos principais de ordenamento do território. Não obstante a continuidade existente entre eles, expressam-se em diferentes geometrias do desenvolvimento: os pontos (polos de crescimento, as linhas (eixos de integração e as zonas (zoneamento ecológico-ecomico. Considerando esses diferentes arranjos territoriais, o artigo busca considerar o papel assumido pelas cidades em cada uma dessas estratégias de desenvolvimento regional, de forma a mostrar mudanças e continuidades que se fazem presentes na história regional recentente da Amazônia

  9. High yield recombinant thermostable α-amylase production using an improved Bacillus licheniformis system

    Directory of Open Access Journals (Sweden)

    Shi Gui-Yang


    Full Text Available Abstract Background Some strains of Bacillus licheniformis have been improved by target-directed screening as well as by classical genetic manipulation and used in commercial thermostable α-amylase and alkaline protease production for over 40 years. Further improvements in production of these enzymes are desirable. Results A new strain of B. licheniformis CBBD302 carrying a recombinant plasmid pHY-amyL for Bacillus licheniformis α-amylase (BLA production was constructed. The combination of target-directed screening and genetic recombination led to an approximately 26-fold improvement of BLA production and export in B. licheniformis. Furthermore, a low-cost fermentation medium containing soybean meal and cottonseed meal for BLA production in shake-flasks and in a 15 liter bioreactor was developed and a BLA concentration of up to 17.6 mg per ml growth medium was attained. Conclusion This production level of BLA by B. licheniformis CBBD302(pHY-amyL is amongst the highest levels in Gram-positive bacteria reported so far.

  10. Identification of an archaeal presenilin-like intramembrane protease. (United States)

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


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

  11. Psicologia Escolar em Rondônia: formação e práticas

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    Iracema Neno Cecilio Tada

    Full Text Available Estudiosos da Psicologia Escolar têm criticado o modelo clínico na formação e atuação dos psicólogos por dificultar a compreensão sobre o funcionamento escolar, sugerindo uma prática pautada na teoria histórico-crítica. Nesta pesquisa, buscou-se conhecer a formação e a atuação dos psicólogos da rede de ensino público de Rondônia. Desenvolveu-se uma pesquisa quantitativa e qualitativa utilizando, como instrumentos para coleta dos dados, questionário, análise documental e diário de campo, com a participação de 38 psicólogos. Os dados mostram que a inserção deste profissional nas escolas é recente em Rondônia. A maioria é ex-aluno da Universidade Federal de Rondônia (UNIR, que, até 2005, possuía enfoque clínico nas disciplinas de Psicologia Escolar, o que parece ter contribuído para a atuação clínica nas escolas. Dado preocupante é a não participação em cursos de formação continuada, contribuindo para uma prática que desconsidera a complexidade do cotidiano escolar e as relações sociais ali constituídas.


    Directory of Open Access Journals (Sweden)

    Felipe Ferreira Vander Velden


    Full Text Available Este artigo discute a introdução de animais domesticados de origem européia em aldeias indígenas na Amazônia, com particular atenção aos grupos em Rondônia, em especial os Karitiana, povo de língua Tupi-Arikém que habita o norte daquele estado. Tendo em vista a história da ocupação do território brasileiro, marcada pela grande narrativa das ‘frentes pastoris’, e a recente expansão – material e ideológica – da pecuária pela floresta amazônica, o texto aponta para várias questões referentes à posição desses animais – sobretudo bois – nos universos simbólicos e materiais das populações indígenas, e sugere possibilidades investigativas que interroguem não apenas sobre as experiências já realizadas com a criação animal em aldeias, mas também os desejos e projetos desenhados para que esta seja implantada.

  13. Tunable protease-activatable virus nanonodes. (United States)

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


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

  14. Identification, recombinant production and partial biochemical characterization of an extracellular cold-active serine-metalloprotease from an Antarctic Pseudomonas isolate

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    Natalia Fullana


    Full Text Available Cold-adapted enzymes are generally derived from psychrophilic microorganisms and have features that make them very attractive for industrial and biotechnological purposes. In this work, we identified a 50 kDa extracellular protease (MP10 from the Antarctic isolate Pseudomonas sp. AU10. The enzyme was produced by recombinant DNA technology, purified using immobilized metal affinity chromatography and partially characterized. MP10 is an alkaline thermosensitive serine-metallo protease with optimal activity at pH 8.0 and 40 ℃, in the presence of 1.5 mM Ca2+. MP10 showed 100% residual activity and stability (up to 60 min when incubated with 7% of non-ionic surfactants (Triton X-100, Tween-80 and Tween-20 and 1.5% of the oxidizing agent hydrogen peroxide. The 3D MP10 structure was predicted and compared with the crystal structure of mesophilic homologous protease produced by Pseudomonas aeruginosa PA01 (reference strain and other proteases, showing similarity in surface area and volume of proteins, but a significantly higher surface pocket area and volume of MP10. The observed differences presumably may explain the enhanced activity of MP10 for substrate binding at low temperatures. These results give insight to the potential use of MP10 in developing new biotechnologically processes active at low to moderate temperatures, probably with focus in the detergent industry.

  15. A experiência com arte na Colônia Juliano Moreira na década de 1950

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    João Henrique Queiroz de Araújo

    Full Text Available Resumo Os trabalhos de Osório César e de Nise da Silveira costumam se destacar quando se investiga a relação entre expressão artística e saberes psi no Brasil em torno da década de 1950. Porém, outras experiências ocorreram no mesmo período, como as atividades de pintura realizadas na Seção de Praxiterapia da Colônia Juliano Moreira, em Jacarepaguá, no município do Rio de Janeiro. A ausência de pesquisas sobre essa experiência suscitou uma busca por documentos que culminou na análise dos números do Boletim da Colônia Juliano Moreira e demais fontes. Os resultados apontam características peculiares da experiência artística na colônia, como o incentivo à cópia e reprodução de fotografias como método terapêutico, o que a diferencia das outras experiências mencionadas.

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

    Directory of Open Access Journals (Sweden)

    Sowdhamini R


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

  17. Desmatamento e a contribuição econômica da floresta na Amazônia

    Directory of Open Access Journals (Sweden)

    Terciane Sabadini Carvalho


    Full Text Available Resumo Este artigo analisa, do ponto de vista econômico, a contribuição da floresta desmatada para a economia da Amazônia Legal entre 2006 e 2011, assim como os efeitos de uma política de controle do desmatamento entre 2012 e 2020. Para isso, utilizou-se um modelo de equilíbrio geral computável (EGC inter-regional, construído para capturar as especificidades e heterogeneidade de 103 microrregiões da Amazônia Legal. As simulações da contribuição econômica do desmatamento para essas regiões permitiriam avaliar o custo relativo de políticas de controle, estimando o valor implícito do desmatamento na dinâmica econômica. Os resultados apontam uma contribuição positiva, embora pequena, do desmatamento para o crescimento econômico da Amazônia de 2006 a 2011. Este resultado é reforçado pelos impactos econômicos de uma política hipotética de controle do desmatamento, que sugerem uma perda apenas marginal sobre o crescimento econômico dessas regiões.

  18. Dataset of cocoa aspartic protease cleavage sites

    Directory of Open Access Journals (Sweden)

    Katharina Janek


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

  19. Correlation between the glycan variations and defibrinogenating activities of acutobin and its recombinant glycoforms.

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    Ying-Ming Wang

    Full Text Available Acutobin isolated from Deinagkistrodon acutus venom has been used to prevent or treat stroke in patients. This defibrinogenating serine protease is a 39 kDa glycoprotein containing terminal disialyl-capped N-glycans. After sialidase treatment, the enzyme showed similar catalytic activities toward chromogenic substrate, and cleaved the Aα chain of fibrinogen as efficiently as the native acutobin did. However, the level of fibrinogen degradation products in mice after i.p.-injection of desialylated-acutobin was significantly lower than the level after acutobin injection, suggesting that the disialyl moieties may improve or prolong the half-life of acutobin. Two recombinant enzymes with identical protein structures and similar amidolytic activities to those of native acutobin were expressed from HEK293T and SW1353 cells and designated as HKATB and SWATB, respectively. Mass spectrometric profiling showed that their glycans differed from those of acutobin. In contrast to acutobin, HKATB cleaved not only the Aα chain but also the Bβ and γ chains of human fibrinogens, while SWATB showed a reduced α-fibrinogenase activity. Non-denaturing deglycosylation of these proteases by peptide N-glycosidase F significantly reduced their fibrinogenolytic activities and thermal stabilities. The in vivo defibrinogenating effect of HKATB was inferior to that of acutobin in mice. Taken together, our results suggest that the conjugated glycans of acutobin are involved in its interaction with fibrinogen, and that the selection of cells optimally expressing efficient glycoforms and further glycosylation engineering are desirable before a recombinant product can replace the native enzyme for clinical use.

  20. Synthesis of glycinamides using protease immobilized magnetic nanoparticles

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    Abha Sahu


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

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

    African Journals Online (AJOL)

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

  2. Optimizing PHB and Protease Production by Box Behnken Design

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    Amro Abd al fattah Amara


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

  3. Appointments on rubber economy and the exploitation of indigenous labor in Rondônia

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    Carlos Alexandre Barros Trubiliano


    Full Text Available The turn of the nineteenth to the twentieth century was marked by the expansion of economic activities in the Amazon region, causing deep socio-spatial impacts. Driven by international rubber demands, sheds, ports and roads were erected. Under the aegis of the progress discourse, thousands of workers were co-opted to labor in the extraction of latex. In the rubber plantations, relations were marked by violence. This essay presents notes on the exploitation of indigenous labor in the formation of rubber plantations in Rondônia. We deal with worker's coping strategies, working conditions and interethnic contact. Our objective was to contribute to the debate about the expansion of capitalist relations in the Amazon region and the impacts of this process on the indigenous populations of Rondônia.

  4. Provocarea Facebook pentru bibliotecile publice din România

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    Octavia-Luciana Madge


    Full Text Available Reţelele sociale şi-au câştigat rapid un loc în viaţa noastră şi în activitatea multor organizaţii şi instituţii publice şi private. La nivelul bibliotecilor, reţelele sociale şi-au demonstrat utilitatea şi rolul în calitate de instrumente de marketing pentru o mai bună comunicare cu utilizatorii. Bibliotecile publice din România au cunoscut în ultimul timp transformări mari în ceea ce priveşte modul de abordare a relaţiei cu utilizatorii şi îmbunătăţirea activităţii lor. Au lansat, de asemenea, o serie de noi servicii pentru a răspunde nevoilor comunităţii pe care o servesc şi pentru a atrage noi utilizatori. Implementarea şi utilizarea de noi aplicaţii precum reţelele de socializare online, mai exact Facebook, a fost una dintre aceste dezvoltări recente. Această lucrare analizează modul în care reţelele sociale sunt utilizate la nivelul bibliotecilor publice din România prin exemplul a trei biblioteci publice mari şi a modului în care aceste biblioteci îşi promovează serviciile prin intermediul reţelei Facebook.

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

    African Journals Online (AJOL)



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

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

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    Celia Torres-Arancivia

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

  7. Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens. (United States)

    Sharma, Vivek; Salwan, Richa; Sharma, Prem N


    In the present study, production of extracellular proteases by Trichoderma harzianum was evaluated based on the relative gene expression and spectrophotometric assay. The fungal isolates were grown in Czapek Dox Broth medium supplemented with deactivated mycelium of plant fungal pathogens such as Fusarium oxysporum, Colletotrichum capsici, Gloeocercospora sorghi, and Colletotrichum truncatum. The maximum protease activity was detected after 48 h of incubation against Colletotrichum spp. Similarly in qRT-PCR, the relative gene expression of four proteases varied from 48 to 96 h against host pathogens in a time-independent manner. Among proteases, statistically significant upregulation of asp, asp, and srp was observed against Colletotrichum spp., followed by F. oxysporum. But in the case of pepM22, maximum upregulation was observed against F. oxysporum. The variation in enzyme assay and qRT-PCR of proteases at different time intervals against various fungal phytopathogens could be due to the limitation of using casein as a substrate for all types of proteases or protease-encoding transcripts selected for qRT-PCR, which may not be true representative of total protease activity.

  8. Economic Methods of Ginger Protease'sextraction and Purification (United States)

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

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

  9. Serine proteases SP1 and SP13 mediate the melanization response of Asian corn borer, Ostrinia furnacalis, against entomopathogenic fungus Beauveria bassiana. (United States)

    Chu, Yuan; Liu, Yang; Shen, Dongxu; Hong, Fang; Wang, Guirong; An, Chunju


    Exposure to entomopathogenic fungi is one approach for insect pest control. Little is known about the immune interactions between fungus and its insect host. Melanization is a prominent immune response in insects in defending against pathogens such as bacteria and fungi. Clip domain serine proteases in insect plasma have been implicated in the activation of prophenoloxidase, a key enzyme in the melanization. The relationship between host melanization and the infection by a fungus needs to be established. We report here that the injection of entomopathogenic fungus Beauveria bassiana induced both melanin synthesis and phenoloxidase activity in its host insect, the Asian corn borer, Ostrinia furnacalis (Guenée). qRT-PCR analysis showed several distinct patterns of expression of 13 clip-domain serine proteases in response to the challenge of fungi, with seven increased, two decreased, and four unchanged. Of special interest among these clip-domain serine protease genes are SP1 and SP13, the orthologs of Manduca sexta HP6 and PAP1 which are involved in the prophenoloxidase activation pathway. Recombinant O. furnacalis SP1 was found to activate proSP13 and induce the phenoloxidase activity in corn borer plasma. Additionally, SP13 was determined to directly cleave prophenoloxidase and therefore act as the prophenoloxidase activating protease. Our work thus reveals a biochemical mechanism in the melanization in corn borer associated with the challenge by B. bassiana injection. These insights could provide valuable information for better understanding the immune responses of Asian corn borer against B. bassiana. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. Crystallization and preliminary crystallographic studies of human kallikrein 7, a serine protease of the multigene kallikrein family

    Energy Technology Data Exchange (ETDEWEB)

    Fernández, Israel S. [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Ständker, Ludger [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain); Hannover Medical School, Center of Pharmacology, 30625 Hannover (Germany); Forssmann, Wolf-Georg [Hannover Medical School, Center of Pharmacology, 30625 Hannover (Germany); Giménez-Gallego, Guillermo; Romero, Antonio, E-mail: [Departamento de Ciencia de Proteínas, Centro de Investigaciones Biológicas-CSIC, Ramiro de Maeztu 9, 28040 Madrid (Spain)


    The cloning, expression, purification and crystallization of recombinant human kallikrein 7, directly synthesized in the active form in E. coli, is described. Diffraction data were collected to 2.8 Å resolution from native crystals. Human kallikreins are a group of serine proteases of high sequence homology whose genes are grouped as a single cluster at chromosome 19. Although the physiological roles of kallikreins are generally still unknown, members of the kallikrein family have been clearly implicated in pathological situations such as cancer and psoriasis. Human kallikrein 7 (hK7) has been shown to be involved in pathological keratinization, psoriasis and ovarian cancer. In order to gain insight into the molecular structure of this protein, hK7 was crystallized after recombinant production in its folded and active form using a periplasmic secretion vector in Escherichia coli. The crystals belonged to the rhombohedral space group H32 and diffracted to 2.8 Å. The phase problem was solved by molecular replacement using the mouse kallikrein-related protein neuropsin. Completion of the model and structure refinement are under way.

  11. Isolation, expression and characterization of a novel dual serine protease inhibitor, OH-TCI, from king cobra venom. (United States)

    He, Ying-Ying; Liu, Shu-Bai; Lee, Wen-Hui; Qian, Jin-Qiao; Zhang, Yun


    Snake venom Kunitz/BPTI members are good tools for understanding of structure-functional relationship between serine proteases and their inhibitors. A novel dual Kunitz/BPTI serine proteinase inhibitor named OH-TCI (trypsin- and chymotrypsin-dual inhibitor from Ophiophagus hannah) was isolated from king cobra venom by three chromatographic steps of gel filtration, trypsin affinity and reverse phase HPLC. OH-TCI is composed of 58 amino acid residues with a molecular mass of 6339Da. Successful expression of OH-TCI was performed as the maltose-binding fusion protein in E. coli DH5alpha. Much different from Oh11-1, the purified native and recombinant OH-TCI both had strong inhibitory activities against trypsin and chymotrypsin although the sequence identity (74.1%) between them is very high. The inhibitor constants (K(i)) of recombinant OH-TCI were 3.91 x 10(-7) and 8.46 x10(-8)M for trypsin and chymotrypsin, respectively. To our knowledge, it was the first report of Kunitz/BPTI serine proteinase inhibitor from snake venom that had equivalent trypsin and chymotrypsin inhibitory activities.

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

    African Journals Online (AJOL)

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

  13. Identification of SlpB, a Cytotoxic Protease from Serratia marcescens. (United States)

    Shanks, Robert M Q; Stella, Nicholas A; Hunt, Kristin M; Brothers, Kimberly M; Zhang, Liang; Thibodeau, Patrick H


    The Gram-negative bacterium and opportunistic pathogen Serratia marcescens causes ocular infections in healthy individuals. Secreted protease activity was characterized from 44 ocular clinical isolates, and a higher frequency of protease-positive strains was observed among keratitis isolates than among conjunctivitis isolates. A positive correlation between protease activity and cytotoxicity to human corneal epithelial cells in vitro was determined. Deletion of prtS in clinical keratitis isolate K904 reduced, but did not eliminate, cytotoxicity and secreted protease production. This indicated that PrtS is necessary for full cytotoxicity to ocular cells and implied the existence of another secreted protease(s) and cytotoxic factors. Bioinformatic analysis of the S. marcescens Db11 genome revealed three additional open reading frames predicted to code for serralysin-like proteases noted here as slpB, slpC, and slpD. Induced expression of prtS and slpB, but not slpC and slpD, in strain PIC3611 rendered the strain cytotoxic to a lung carcinoma cell line; however, only prtS induction was sufficient for cytotoxicity to a corneal cell line. Strain K904 with deletion of both prtS and slpB genes was defective in secreted protease activity and cytotoxicity to human cell lines. PAGE analysis suggests that SlpB is produced at lower levels than PrtS. Purified SlpB demonstrated calcium-dependent and AprI-inhibited protease activity and cytotoxicity to airway and ocular cell lines in vitro. Lastly, genetic analysis indicated that the type I secretion system gene, lipD, is required for SlpB secretion. These genetic data introduce SlpB as a new cytotoxic protease from S. marcescens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  14. Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities. (United States)

    Qian, Ye-Qing; Li, Ye; Yang, Fan; Yu, Yan-Qin; Yang, Jin-Shu; Yang, Wei-Jun


    Kazal-type inhibitors (KPIs) play important roles in many biological and physiological processes, such as blood clotting, the immune response and reproduction. In the present study, two male reproductive tract KPIs, termed Man-KPI and Ers-KPI, were identified in Macrobrachium nipponense and Eriocheir sinensis, respectively. The inhibitory activities of recombinant Man-KPI and Ers-KPI against chymotrypsin, elastase, trypsin and thrombin were determined. The results showed that both of them strongly inhibit chymotrypsin and elastase. Kinetic studies were performed to elucidate their inhibition mechanism. Furthermore, individual domains were also expressed to learn further which domain contributes to the inhibitory activities of intact KPIs. Only Man-KPI_domain3 is active in the inhibition of chymotrypsin and elastase. Meanwhile, Ers-KPI_domain2 and 3 are responsible for inhibition of chymotrypsin, and Ers-KPI_domains2, 3 and 4 are responsible for the inhibition of elastase. Meanwhile, the inhibitory activities of these two KPIs toward Macrobrachium rosenbergii, M. nipponense and E. sinensis sperm were compared with that of the Kazal-type peptidase inhibitor (MRPINK) characterized from the M. rosenbergii reproductive tract in a previous study. The results demonstrated that KPIs can completely inhibit the gelatinolytic activities of sperm proteases from their own species, while different levels of cross-inhibition were observed between KPI and proteases from different species. These results may provide new perspective to further clarify the mechanism of KPI-proteases interaction in the male reproductive system. Crown Copyright © 2011. Published by Elsevier Ltd. All rights reserved.

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

    African Journals Online (AJOL)



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

  16. Current and Novel Inhibitors of HIV Protease

    Czech Academy of Sciences Publication Activity Database

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


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

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

    Directory of Open Access Journals (Sweden)

    Sonia eVerma


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

  18. Single-step affinity and cost-effective purification of recombinant proteins using the Sepharose-binding lectin-tag from the mushroom Laetiporus sulphureus as fusion partner. (United States)

    Li, Xiao-Jing; Liu, Jin-Ling; Gao, Dong-Sheng; Wan, Wen-Yan; Yang, Xia; Li, Yong-Tao; Chang, Hong-Tao; Chen, Lu; Wang, Chuan-Qing; Zhao, Jun


    Previous research showed that a lectin from the mushroom Laetiporus sulphureus, designed LSL, bound to Sepharose and could be eluted by lactose. In this study, by taking advantage of the strong affinity of LSL-tag for Sepharose, we developed a single-step purification method for LSL-tagged fusion proteins. We utilized unmodified Sepharose-4B as a specific adsorbent and 0.2 M lactose solution as an elution buffer. Fusion proteins of LSL-tag and porcine circovirus capsid protein, designated LSL-Cap was recovered with purity of 90 ± 4%, and yield of 87 ± 3% from crude extract of recombinant Escherichia coli. To enable the remove of LSL-tag, tobacco etch virus (TEV) protease recognition sequence was placed downstream of LSL-tag in the expression vector, and LSL-tagged TEV protease, designated LSL-TEV, was also expressed in E. coli., and was recovered with purity of 82 ± 5%, and yield of 85 ± 2% from crude extract of recombinant E. coli. After digestion of LSL-tagged recombinant proteins with LSL-TEV, the LSL tag and LSL-TEV can be easily removed by passing the digested products through the Sepharose column. It is of worthy noting that the Sepharose can be reused after washing with PBS. The LSL affinity purification method enables rapid and inexpensive purification of LSL-tagged fusion proteins and scale-up production of native proteins. Copyright © 2015 Elsevier Inc. All rights reserved.

  19. Characterization of the Mamestra configurata (Lepidoptera: Noctuidae) larval midgut protease complement and adaptation to feeding on artificial diet, Brassica species, and protease inhibitor. (United States)

    Erlandson, Martin A; Hegedus, Dwayne D; Baldwin, Douglas; Noakes, Amy; Toprak, Umut


    The midgut protease profiles from 5th instar Mamestra configurata larvae fed various diets (standard artificial diet, low protein diet, low protein diet with soybean trypsin inhibitor [SBTI], or Brassica napus) were characterized by one-dimensional enzymography in gelatin gels. The gut protease profile of larvae fed B. napus possessed protease activities of molecular masses of approximately 33 and 55 kDa, which were not present in the guts of larvae fed artificial diet. Similarly, larvae fed artificial diet had protease activities of molecular masses of approximately 21, 30, and 100 kDa that were absent in larvae fed B. napus. Protease profiles changed within 12 to 24 h after switching larvae from artificial diet to plant diet and vice versa. The gut protease profiles from larvae fed various other brassicaceous species and lines having different secondary metabolite profiles did not differ despite significant differences in larval growth rates on the different host plants. Genes encoding putative digestive proteolytic enzymes, including four carboxypeptidases, five aminopeptidases, and 48 serine proteases, were identified in cDNA libraries from 4th instar M. configurata midgut tissue. Many of the protease-encoding genes were expressed at similar levels on all diets; however, three chymoptrypsin-like genes (McSP23, McSP27, and McSP37) were expressed at much higher levels on standard artificial diet and diet containing SBTI as was the trypsin-like gene McSP34. The expression of the trypsin-like gene McSP50 was highest on B. napus. The adaptation of M. configurata digestive biochemistry to different diets is discussed in the context of the flexibility of polyphagous insects to changing diet sources.

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

    Directory of Open Access Journals (Sweden)

    E. V. Маtseliukh


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

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

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin


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

  2. Antibody proteases: induction of catalytic response. (United States)

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


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

  3. Characterization of detergent compatible protease from halophilic Virgibacillus sp. CD6. (United States)

    Lam, Ming Quan; Nik Mut, Nik Nurhidayu; Thevarajoo, Suganthi; Chen, Sye Jinn; Selvaratnam, Chitra; Hussin, Huszalina; Jamaluddin, Haryati; Chong, Chun Shiong


    A halophilic bacterium, Virgibacillus sp. strain CD6, was isolated from salted fish and its extracellular protease was characterized. Protease production was found to be highest when yeast extract was used as nitrogen source for growth. The protease exhibited stability at wide range of salt concentration (0-12.5%, w/v), temperatures (20-60 °C), and pH (4-10) with maximum activity at 10.0% (w/v) NaCl, 60 °C, pH 7 and 10, indicating its polyextremophilicity. The protease activity was enhanced in the presence of Mg 2+ , Mn 2+ , Cd 2+ , and Al 3+ (107-122% relative activity), and with retention of activity > 80% for all of other metal ions examined (K + , Ca 2+ , Cu 2+ , Co 2+ , Ni 2+ , Zn 2+ , and Fe 3+ ). Both PMSF and EDTA inhibited protease activity, denoting serine protease and metalloprotease properties, respectively. High stability (> 70%) was demonstrated in the presence of organic solvents and detergent constituents, and the extracellular protease from strain CD6 was also found to be compatible in commercial detergents. Proteinaceous stain removal efficacy revealed that crude protease of strain CD6 could significantly enhance the performance of commercial detergent. The protease from Virgibacillus sp. strain CD6 could serve as a promising alternative for various applications, especially in detergent industry.

  4. Functional Implications of Domain Organization Within Prokaryotic Rhomboid Proteases. (United States)

    Panigrahi, Rashmi; Lemieux, M Joanne


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

  5. The Degradome database: mammalian proteases and diseases of proteolysis. (United States)

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


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

  6. New tick records in Rondônia, Western Brazilian Amazon


    Labruna,Marcelo Bahia; Barbieri,Fábio Silva; Martins,Thiago Fernandes; Brito,Luciana Gatto; Ribeiro,Francisco Dimas Sales


    In the present study, we provide new tick records from Vilhena Municipality, in the Southeast of the State of Rondônia, Northern Brazil. Ticks collected from a capybara, Hydrochoerus hydrochaeris (Linnaeus), were identified as Amblyomma romitii Tonelli-Rondelli (1 female), and Amblyomma sp. (1 larva). Ticks collected from a harpy eagle, Harpia harpyja (Linnaeus), were identified as Amblyomma cajennense (Fabricius) (16 nymphs) and Haemaphysalis juxtakochi Cooley (1 nymph). Ticks collected from...

  7. Prototype Packages for Managing and Animating Longitudinal Network Data: dynamicnetwork and rSoNIA

    Directory of Open Access Journals (Sweden)

    Skye Bender-deMoll


    Full Text Available Work with longitudinal network survey data and the dynamic network outputs of the statnet ERGMs has demonstrated the need for consistent frameworks and data structures for expressing, storing, and manipulating information about networks that change in time. Motivated by our requirements for exchanging data among researchers and various analysis and visualization processes, we have created an R package dynamicnetwork that builds upon previous work in the network, statnet and sna packages and provides a limited functional implementation. This paper discusses design issues and considerations, describes classes and forms of dynamic data, and works through several examples to demonstrate the utility of the package. The functionality of the rSoNIA package that uses dynamicnetwork to exchange data with the Social Network Image Animator (SoNIA software to create animated movies of changing networks from within R is also demonstrated.

  8. 21 CFR 184.1027 - Mixed carbohydrase and protease enzyme product. (United States)


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

  9. Recent observations on the sand fly (Diptera: Psychodidae fauna of the State of Rondônia, Western Amazônia, Brazil: the importance of Psychdopygus davisi as a vector of zoonotic cutaneous leishmaniasis

    Directory of Open Access Journals (Sweden)

    Gil Luis HS


    Full Text Available Sand flies were collected in the central region of the state of Rondônia (W 64º30' to 63º00' and S 10º00'to 11º00' using Shannon and CDC light traps from October 1997 to August 2000. A total of 85,850 specimens representing 78 named species were captured. Of these 14 were new records for Rondônia. The proportion of males/females was 1/1.131. Trypanosomatids, that are presently being identified, were detected in 11 species. Leishmania (Viannia naiffi was recorded from Psychodopygus davisi and P. hirsutus. In the present study the dominant species was P. davisi (39.6% followed by Lutzomyia whitmani (13.1%, P. carrerai (11.6%, and P. hirsutus (10.2%. The importance of P. davisi as a vector of zoonotic cutaneous leishmaniasis is discussed.

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

    DEFF Research Database (Denmark)

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


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

  11. Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

    International Nuclear Information System (INIS)

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


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

  12. Aplicação do método da carga máxima total diária (CMTD para a amônia no Rio Atibaia, região de Campinas/Paulínia - SP Application of ammonia total maximum daily load (TMDL to Atibaia River, Campinas/Paulínia region - São Paulo state

    Directory of Open Access Journals (Sweden)

    Gilberto Silvério da Silva


    Full Text Available Neste estudo foram avaliadas a capacidade de suporte e o estado de degradação do Rio Atibaia, considerando a ameaça para a vida aquática pela presença da Amônia, a qual representa um dos principais riscos às comunidades aquáticas no Rio Atibaia. Com este objetivo foi aplicado o método da Carga Máxima Total Diária (CMTD, da Agência de Proteção Ambiental dos Estados Unidos (EPA. Os resultados revelaram que as cargas de Amônia aumentavam progressivamente ao longo do Rio Atibaia, principalmente devido às fontes pontuais. As cargas de Amônia diárias assumiram valores de 30 a 5000 kg NH3. A capacidade de suporte das águas Rio Atibaia, para proteger a vida aquática contra os efeitos tóxicos da Amônia, tem sido violadas em trechos próximos à sua foz. A degradação dessas águas foi mais intensa na estação seca. Este trabalho mostrou que o esgoto doméstico não-tratado de uma população aproximada de 250 mil habitantes da cidade de Campinas, via Ribeirão Anhumas, é a principal fonte de Amônia na bacia do Rio Atibaia, apesar do grande número de indústrias ali presentes.This study evaluated the tolerance capacity and the impairment state of the Atibaia River, considering the threat to aquatic life by the presence of Ammonia, which represents one of the main risks to the aquatic communities in the Atibaia River. With this aim, the method Total Maximum Daily Load (TMDL, from the United States Environmental Protection Agency (EPA, was applied. The results revealed that the Ammonia loads increased progressively through the Atibaia River, especially due to the point sources. The daily Ammonia loads assumed values that ranged from 30 to 5000 kg NH3. The tolerance capacity of the waters of the Atibaia River, to protect aquatic life against the toxic effects of the Ammonia, has been violated in reaches near its mouth. The impairment of these waters was more intense during the dry season. This study showed that the domestic sewer

  13. Análise da adequação de eventos de precipitação extrema na Amazônia Ocidental em modelos estáticos: Rondônia

    Directory of Open Access Journals (Sweden)

    Vinicius Alexandre Sikora de Souza


    Full Text Available Este estudo objetivou estimar a função Intensidade-Duração-Frequência (IDF de eventos pluviométricos extremos a partir dos dados de precipitação das estações pluviométricas instaladas no estado de Rondônia, de modo que posteriormente tais informações possam ser utilizadas no dimensionamento de obras hidráulicas. Utilizou-se 41 estações pluviométricas com séries históricas acima de 10 anos, disponibilizadas pela Agência Nacional de Águas (ANA. Essas séries passaram inicialmente pelo teste de aderência Kolmogorov-Smirnov (KS, a fim de verificar o ajuste das mesmas as distribuições: Normal, Log-Normal, Exponencial, Gama, Gumbel, Weibull e Logística. O trabalho denotou que o teste de aderência Kolmogorov-Smirnov de forma geral forneceu uma expressiva aceitação na maioria das distribuições estatística testadas. Analysis of fitness for extreme rainfall events in western amazon in static models: state Rondônia ABSTRACT: This study aimed to estimate the Intensity - Duration - Frequency (IDF function extreme rainfall events from the data of precipitation of rainfall stations located in the State of Rondônia, so that such information can be later used in hydraulic structures. We used 41 rainfall stations with historical series over 10 years, provided by the National Water Agency (ANA. These series originally started by adherence Kolmogorov -Smirnov (KS in order to check the fit of the same distributions: Normal, Log- Normal, Exponential, Gamma, Gumbel, Weibull and Logistics. Work denoted that the Kolmogorov - Smirnov test of adherence generally provided a significant acceptance in most of the tested statistical distributions.

  14. Malaria prevalence in Nias District, North Sumatra Province, Indonesia

    Directory of Open Access Journals (Sweden)

    Laowo Idaman


    Full Text Available Abstract Background The Nias district of the North Sumatra Province of Indonesia has long been known to be endemic for malaria. Following the economic crisis at the end of 1998 and the subsequent tsunami and earthquake, in December 2004 and March 2005, respectively, the malaria control programme in the area deteriorated. The present study aims to provide baseline data for the establishment of a suitable malaria control programme in the area and to analyse the frequency distribution of drug resistance alleles associated with resistance to chloroquine and sulphadoxine-pyrimethamine. Methods Malariometric and entomology surveys were performed in three subdistricts. Thin and thick blood smears were stained with Giemsa and examined under binocular light microscopy. Blood blots on filter paper were also prepared for isolation of parasite and host DNA to be used for molecular analysis of band 3 (SAO, pfcrt, pfmdr1, dhfr, and dhps. In addition, haemoglobin measurement was performed in the second and third surveys for the subjects less than 10 years old. Results Results of the three surveys revealed an average slide positivity rate of 8.13%, with a relatively higher rate in certain foci. Host genetic analysis, to identify the Band 3 deletion associated with Southeast Asian Ovalocytosis (SAO, revealed an overall frequency of 1.0% among the 1,484 samples examined. One hundred six Plasmodium falciparum isolates from three sub-districts were successfully analysed. Alleles of the dhfr and dhps genes associated with resistance to sulphadoxine-pyrimethamine, dhfr C59R and S108N, and dhps A437G and K540E, were present at frequencies of 52.2%, 82.5%, 1.18% and 1.18%, respectively. The pfmdr1 alleles N86Y and N1042D, putatively associated with mefloquine resistance, were present at 31.4% and 2%, respectively. All but one sample carried the pfcrt 76T allele associated with chloroquine resistance. Entomologic surveys identified three potential anopheline vectors in

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

    Directory of Open Access Journals (Sweden)

    Ladislau C. Kovari


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

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

    Directory of Open Access Journals (Sweden)

    Huan Zhang


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

  17. Kinetics Study of Extracellular Detergent Stable Alkaline Protease from Rhizopus oryzae

    Directory of Open Access Journals (Sweden)

    Zareena Mushtaq


    Full Text Available In this study, extracellular alkaline protease was produced from Rhizopus oryzae in submerged fermentation using dairy waste (whey as a substrate. Fermentation kinetics was studied and various parameters were optimized. The strain produced maximum protease at initial medium pH of 6.0 medium depth of 26 mm, inoculum size of 2% at incubation temperature of 35ºC for 168 h of fermentation. Alkaline protease was purified to homogeneity by ammonium sulphate fractionation followed by sephadex G-100 chromatography. The molecular mass of alkaline protease was 69 kDa determined by 10% SDS-PAGE. The optimum pH and temperature of alkaline protease was 9.0 and 40ºC, respectively. Metal profile of the enzyme showed that the enzyme was non-metallic in nature. The Km , Kcat , Vmax and Kcat/Km values of purified protease were 7.0 mg/mL, 3.8 x102S-1, 54.30 µmol/min and 54.28 s-1mg -1.mL respectively, using casein as substrate. The purified alkaline protease had stability with commercial detergents.

  18. Reverse zymography alone does not confirm presence of a protease inhibitor. (United States)

    Dutta, Sangita; Bhattacharyya, Debasish


    Reverse zymography is applied for identification and semi-quantification of protease inhibitors that are of protein in nature. However, a protein that shows band in reverse zymography against a protease used for digestion of the gel need not be an inhibitor; it might be resistant to degradation by the protease. We demonstrate that in reverse zymography, avidin, streptavidin and the leaf extract of Catharanthus roseus behave like inhibitors of proteases like papain, ficin, bromelain extracts from pineapple leaf, stem and fruit and trypsin. Still, they do not act as inhibitors of those proteases when enzyme assays were done in solution. In reverse zymography, the extract of pineapple crown leaf shows two major inhibitor bands against its own proteases. Identification of these proteins from sequences derived from MALDI TOF MS analysis indicated that they are fruit and stem bromelains. Avidin, streptavidin and bromelains are 'kinetically stable proteins' that are usually resistant to proteolysis. Thus, it is recommended that identification of an inhibitor of a protease by reverse zymography should be supported by independent assay methods for confirmation.

  19. Plant proteases for bioactive peptides release: A review. (United States)

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


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

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

    African Journals Online (AJOL)



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

  1. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    DEFF Research Database (Denmark)

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt


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

  2. The non-death role of metacaspase proteases

    International Nuclear Information System (INIS)

    Shrestha, Amit; Megeney, Lynn A.


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

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

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    Michael Schauperl

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

  4. Functional and structural characterization of recombinant dermcidin-1L, a human antimicrobial peptide

    International Nuclear Information System (INIS)

    Lai Yuping; Peng Yifei; Zuo Yi; Li Jun; Huang Jing; Wang Linfa; Wu Zirong


    Antimicrobial peptides from human skin are an important component of the innate immune response and play a key role as a first line of defense against infections. One such peptide is the recently discovered dermcidin-1L. To better understand its mechanism and to further investigate its antimicrobial spectrum, recombinant dermcidin-1L was expressed in Escherichia coli as a fusion protein and purified by affinity chromatography. The fusion protein was cleaved by factor Xa protease to produce recombinant dermcidin-1L. Antimicrobial and hemolytic assays demonstrated that dermcidin-1L displayed microbicidal activity against several opportunistic nosocomial pathogens, but no hemolytic activity against human erythrocytes even at concentrations up to 100 μM. Structural studies performed by circular dichroism spectroscopy indicated that the secondary structure of dermcidin-1L was very flexible, and both α-helix and β-sheet structures might be required for the antimicrobial activity. Our results confirmed previous findings indicating that dermcidin-1L could have promising therapeutic potentials and shed new light on the structure-function relationship of dermcidin-1L

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

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


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

  6. Overexpression of Aspergillus tubingensis faeA in protease-deficient Aspergillus niger enables ferulic acid production from plant material. (United States)

    Zwane, Eunice N; Rose, Shaunita H; van Zyl, Willem H; Rumbold, Karl; Viljoen-Bloom, Marinda


    The production of ferulic acid esterase involved in the release of ferulic acid side groups from xylan was investigated in strains of Aspergillus tubingensis, Aspergillus carneus, Aspergillus niger and Rhizopus oryzae. The highest activity on triticale bran as sole carbon source was observed with the A. tubingensis T8.4 strain, which produced a type A ferulic acid esterase active against methyl p-coumarate, methyl ferulate and methyl sinapate. The activity of the A. tubingensis ferulic acid esterase (AtFAEA) was inhibited twofold by glucose and induced twofold in the presence of maize bran. An initial accumulation of endoglucanase was followed by the production of endoxylanase, suggesting a combined action with ferulic acid esterase on maize bran. A genomic copy of the A. tubingensis faeA gene was cloned and expressed in A. niger D15#26 under the control of the A. niger gpd promoter. The recombinant strain has reduced protease activity and does not acidify the media, therefore promoting high-level expression of recombinant enzymes. It produced 13.5 U/ml FAEA after 5 days on autoclaved maize bran as sole carbon source, which was threefold higher than for the A. tubingensis donor strain. The recombinant AtFAEA was able to extract 50 % of the available ferulic acid from non-pretreated maize bran, making this enzyme suitable for the biological production of ferulic acid from lignocellulosic plant material.

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

    African Journals Online (AJOL)

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

  8. m-AAA proteases, mitochondrial calcium homeostasis and neurodegeneration. (United States)

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


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

  9. Constitutive non-inducible expression of the Arabidopsis thaliana Nia 2 gene in two nitrate reductase mutants of Nicotiana plumbaginifolia. (United States)

    Kaye, C; Crawford, N M; Malmberg, R L


    We have isolated a haploid cell line of N. plumbaginifolia, hNP 588, that is constitutive and not inducible for nitrate reductase. Nitrate reductase mutants were isolated from hNP 588 protoplasts upon UV irradiation. Two of these nitrate reductase-deficient cell lines, nia 3 and nia 25, neither of which contained any detectable nitrate reductase activity, were selected for complementation studies. A cloned Arabidopsis thaliana nitrate reductase gene Nia 2 was introduced into each of the two mutants resulting in 56 independent kanamycin-resistant cell lines. Thirty of the 56 kanamycin-resistant cell lines were able to grow on nitrate as the sole nitrogen source. Eight of these were further analyzed for nitrate reductase enzyme activity and nitrate reductase mRNA production. All eight lines had detectable nitrate reductase activity ranging from 7% to 150% of wild-type hNP 588 callus. The enzyme activity levels were not influenced by the nitrogen source in the medium. The eight lines examined expressed a constitutive, non-inducible 3.2 kb mRNA species that was not present in untransformed controls.

  10. Production, Partial Purification and Characterization of Protease From Irradiated Streptomyces Spp

    International Nuclear Information System (INIS)

    Botros, H.W.; Ahmed, A.S.


    Production and partial purification of protease by the irradiated Streptomyces spp. was the aim of this study. Streptomyces spp. was allowed to grow in culture broth of 4% shrimp shells for purpose of inducing protease enzymes. Optimal conditions for protease production were 30 degree C, 0.3 kGy, ph 7, 5x10 4 /ml inoculum size and 7 days incubation period. Protease was purified by 80% ammonium sulphate saturation which exhibited 8.7 U/ml enzyme activity. Column chromatography using sephadex G-200 exerted 23.3 U/ml enzyme activity from pooled fraction (13-16). The molecular mass of protease was determined to be 39 kDa by SDS-PAGE. The enzyme was more stable over a wide range of ph 6-8 and temperature up to 40 degree C. The produced protease was activated by Ca, Mn and FeCl 2 and completely inhibited by ethylene-diamin tetraacetic acid (EDTA) at concentration of 1000 μg/ml

  11. Proteases from Latex of Euphorbia spp. and Its Application on Milk Clot Formation

    Directory of Open Access Journals (Sweden)

    Fidia Fibriana


    Full Text Available Crude proteases were extracted from Euphorbiaceae family, i.e. E. milii var imperata, E. trigona, and E. maculata. Among those three crude proteases, the activity of protease from E. trigona was the highest (812.50 U/ml, whereas E. milii and E. maculata crude proteases activity were 298.60 U/ml and 95.80 U/ml, respectively. E. maculata protein concentration was the highest among those three crude enzymes (1.206 mg/ml. The optimum pH and temperature of the enzymes were pH 7.0, pH 6.0, pH 6.5 and 60 °C, 50 °C, and 50 °C, respectively. Crude protease from E. milii var imperata, E. trigona, and E. maculata retained proteolytic activity over a wide range of pH (5.0–9.0 and temperature (up to 65 °C with casein as substrate. All crude proteases showed milk clotting activity ranged from 0.58 U/ml to 1.01 U/ml. Thus, these crude proteases are potential to be applied in dairy industries. However, further study on enzyme purification and characterization are necessary to obtain high purity of proteases before its application.Protease kasar berhasil diekstrak dari tanaman family Euphorbiaceae, yaitu E. milii var imperata, E. trigona, dan E. maculata. Diantara ketiga protease tersebut, aktivitas protease tertinggi diperoleh dari E. trigona (812,50 U/ml, sedangkan aktivitas protease dari E. milii dan E. maculata adalah 298,60 U/ml dan 95,80 U/ml, berturut-turut. Konsentrasi total protein tertinggi terdapat pada protease kasar E. maculata (1,206 mg/ml. pH dan suhu optimum ketiga enzim tersebut adalah pH 7.0, pH 6.0, pH 6.5 dan suhu 60 °C, 50 °C, and 50 °C, berturut-turut. Protease kasar dari E. milii var imperata, E. trigona, dan E. maculata menunjukkan aktivitas proteolitik pada rentang pH 5.0–9.0 dan rentang suhu sampai 65 °C menggunakan kasein sebagai substrat. Semua protease kasar menunjukkan aktivitas penggumpalan susu dengan rentang dari 0,58 U/ml sampai 1,01 U/ml. Berdasarkan hasil yang diperoleh, protease kasar dari ketiga jenis tanaman ini

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

    Directory of Open Access Journals (Sweden)

    Yu-Yin Li

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

  13. O Programa de Aquisição de Alimentos Na Amazônia Legal Brasileira

    Directory of Open Access Journals (Sweden)

    Alessandro Antônio Cavallari


    Full Text Available A presente revisão bibliográfica possibilita uma análise da evolução do Programa Aquisição de Alimentos nos primeiros sete anos de sua instituição na região da Amazônia legal brasileira, a partir da quantidade de municípios atendidos por Unidade Federativa que compõe essa região, através de dados obtidos pela CONAB. No primeiro ano de implantação do programa, apenas os estados do Acre, Rondônia, Amazonas e Maranhão possuíam municípios atendidos por essa política de incentivo a agricultura familiar. Com o decorrer dos anos, algumas mudanças foram constatadas, principalmente com a adesão de novos municípios ao programa, aumentando a abrangência de atendimento do PAA na região. No ano de 2009 foi verificada uma situação atípica, onde foi observada a redução na quantidade de municípios vinculados ao programa, a partir de então houve um crescimento generalizado no atendimento em todos os Estados que compõe a Amazônia legal brasileira.O programa se encontra em fase de consolidação, o que possibilita o crescimento e abrangência no atendimento a agricultura familiar da região Amazônica.

  14. Cold denaturation of the HIV-1 protease monomer

    DEFF Research Database (Denmark)

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


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

  15. Expression and Purification of Recombinant Proteins in Escherichia coli with a His6 or Dual His6-MBP Tag. (United States)

    Raran-Kurussi, Sreejith; Waugh, David S


    Rapid advances in bioengineering and biotechnology over the past three decades have greatly facilitated the production of recombinant proteins in Escherichia coli. Affinity-based methods that employ protein or peptide based tags for protein purification have been instrumental in this progress. Yet insolubility of recombinant proteins in E. coli remains a persistent problem. One way around this problem is to fuse an aggregation-prone protein to a highly soluble partner. E. coli maltose-binding protein (MBP) is widely acknowledged as a highly effective solubilizing agent. In this chapter, we describe how to construct either a His 6 - or a dual His 6 -MBP tagged fusion protein by Gateway ® recombinational cloning and how to evaluate their yield and solubility. We also describe a simple and rapid procedure to test the solubility of proteins after removing their N-terminal fusion tags by tobacco etch virus (TEV) protease digestion. The choice of whether to use a His 6 tag or a His 6 -MBP tag can be made on the basis of this solubility test.

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

    Directory of Open Access Journals (Sweden)

    Chen Chaoping


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

  17. Two-Dimensional Zymography of Proteases from Steatotic Duck Liver. (United States)

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


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

  18. Molecular assembly of recombinant chicken type II collagen in the yeast Pichia pastoris. (United States)

    Xi, Caixia; Liu, Nan; Liang, Fei; Zhao, Xiao; Long, Juan; Yuan, Fang; Yun, Song; Sun, Yuying; Xi, Yongzhi


    Effective treatment of rheumatoid arthritis can be mediated by native chicken type II collagen (nCCII), recombinant peptide containing nCCII tolerogenic epitopes (CTEs), or a therapeutic DNA vaccine encoding the full-length CCOL2A1 cDNA. As recombinant CCII (rCCII) might avoid potential pathogenic virus contamination during nCCII preparation or chromosomal integration and oncogene activation associated with DNA vaccines, here we evaluated the importance of propeptide and telopeptide domains on rCCII triple helix molecular assembly. We constructed pC- and pN-procollagen (without N- or Cpropeptides, respectively) as well as CTEs located in the triple helical domain lacking both propeptides and telopeptides, and expressed these in yeast Pichia pastoris host strain GS115 (his4, Mut + ) simultaneously with recombinant chicken prolyl-4-hydroxylase α and β subunits. Both pC- and pN-procollagen monomers accumulated inside P. pastoris cells, whereas CTE was assembled into homotrimers with stable conformation and secreted into the supernatants, suggesting that the large molecular weight pC-or pN-procollagens were retained within the endoplasmic reticulum whereas the smaller CTEs proceeded through the secretory pathway. Furthermore, resulting recombinant chicken type II collagen pCα1(II) can induced collagen-induced arthritis (CIA) rat model, which seems to be as effective as the current standard nCCII. Notably, protease digestion assays showed that rCCII could assemble in the absence of C- and N-propeptides or telopeptides. These findings provide new insights into the minimal structural requirements for rCCII expression and folding.

  19. Utilizing protein-lean coproducts from corn containing recombinant pharmaceutical proteins for ethanol production. (United States)

    Paraman, Ilankovan; Moeller, Lorena; Scott, M Paul; Wang, Kan; Glatz, Charles E; Johnson, Lawrence A


    Protein-lean fractions of corn (maize) containing recombinant (r) pharmaceutical proteins were evaluated as a potential feedstock to produce fuel ethanol. The levels of residual r-proteins in the coproduct, distillers dry grains with solubles (DDGS), were determined. Transgenic corn lines containing recombinant green fluorescence protein (r-GFP) and a recombinant subunit vaccine of Escherichia coli enterotoxin (r-LTB), primarily expressed in endosperm, and another two corn lines containing recombinant human collagen (r-CIα1) and r-GFP, primarily expressed in germ, were used as model systems. The kernels were either ground and used for fermentation or dry fractionated to recover germ-rich fractions prior to grinding for fermentation. The finished beers of whole ground kernels and r-protein-spent endosperm solids contained 127-139 and 138-155 g/L ethanol concentrations, respectively. The ethanol levels did not differ among transgenic and normal corn feedstocks, indicating the residual r-proteins did not negatively affect ethanol production. r-Protein extraction and germ removal also did not negatively affect fermentation of the remaining mass. Most r-proteins were inactivated during the mashing process used to prepare corn for fermentation. No functionally active r-GFP or r-LTB proteins were found after fermentation of the r-protein-spent solids; however, a small quantity of residual r-CIα1 was detected in DDGS, indicating that the safety of DDGS produced from transgenic grain for r-protein production needs to be evaluated for each event. Protease treatment during fermentation completely hydrolyzed the residual r-CIα1, and no residual r-proteins were detectable in DDGS.

  20. Associação entre sintomas de insônia e artrite reumatóide em idosos

    Directory of Open Access Journals (Sweden)

    Denise Cuoghi de Carvalho Verissimo Freitas


    Full Text Available Esse estudo objetivou avaliar sintomas de insônia em idosos residentes na comunidade e sua associação com a artrite reumatóide. Estudo descritivo, de corte transversal, recorte do projeto multicêntrico Fragilidade em Idosos Brasileiros . Foram avaliados 689 idosos (68,9% mulheres, média de idade 72,2 anos utilizando-se questionário sociodemográfico e sobre condições clínicas, questões sobre sintomas de insônia (Perfil de Saúde de Nottingham e teste de rastreio para alterações cognitivas (Mini-exame do Estado Mental. Utilizou-se análise descritiva e análise de regressão logística múltipla, com nível de significância de 5%. A associação entre sintomas de insônia e artrite reumatóide manteve-se significativa na análise multivariada; outros fatores associados foram avaliação subjetiva da saúde muito ruim, não ser aposentado e apresentar depressão. Os profissionais da área da saúde devem investigar cuidadosamente a qualidade do sono noturno dos idosos acometidos por artrite reumatóide, dada sua elevada prevalência nessa população.

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

    Directory of Open Access Journals (Sweden)

    Kuo-Feng Weng


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

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

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

  4. Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS. (United States)

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


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

  5. Proteases in Escherichia coli and Staphylococcus aureus confer reduced susceptibility to lactoferricin B. (United States)

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


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

  6. Damages caused by Bipolaris maydis in Panicum maximum cv. Tanzânia Dano causado por Bipolaris maydis em Panicum maximum cv. Tanzânia

    Directory of Open Access Journals (Sweden)

    Gilmar Franzener


    Full Text Available The aim of this research was to evaluate the damage caused by Bipolaris maydis in the quantity and quality of the Tanzania grass production. Tanzania grass plants were cultivated in pots of PVC tubes (50 cm of height x 15 cm of diameter containing a mixture of soil and sand (3:1. The plants were inoculated with spore pathogen suspension containing 102, 104 and 106 conidia/mL to obtain different levels of disease. Severity and number of tillers were evaluated weekly. After four weeks, it were evaluated the weight of the fresh matter of leaves (FM, percentage of dry matter (DM, crude protein (CP, neutral detergent fiber (NDF and acid detergent fiber (ADF. The disease reduced significantly the plant tillering and FM after the second evaluation (P O objetivo deste trabalho foi avaliar o dano causado por Bipolaris maydis (helmintosporiose na quantidade e qualidade da produção do capim Tanzânia. Plantas de capim Tanzânia foram cultivadas em vasos de tubos de PVC (50 cm de altura x 15 cm de diâmetro contendo mistura solo/areia (3:1. As plantas foram inoculadas com suspensões de esporos do patógeno com diferentes concentrações (102, 104 e 106 conídios/mL, visando obter gradiente de severidade de manchas foliares. Foram realizadas avaliações semanais da severidade e do número de perfilhos. Após quatro semanas avaliou-se o peso da matéria fresca de folhas (MF, porcentagem de matéria seca (PMS, de proteína bruta (PB, de fibra em detergente neutro (FDN e de fibra em detergente ácido (FDA. A doença reduziu significativamente o perfilhamento e MF a partir da segunda avaliação (P < 0,05. Não houve correlação significativa entre as variáveis PMS, FDA e área abaixo da curva de progresso da doença (AACPD. No entanto, houve correlação positiva (P < 0,01 e negativa (P < 0 ,05 entre PB e FDN, respectivamente, com a AACPD, como possível resultado da atividade do patógeno. Estes resultados indicam que B. maydis inibe o desenvolvimento do

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

    Directory of Open Access Journals (Sweden)

    Yasar Demir


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


    Directory of Open Access Journals (Sweden)

    Vinícius Machado Rocha


    Full Text Available Este artigo traz uma perspectiva histórica da evolução do conhecimento sobre a reciclagem de precipitação e fornece uma visão crítica do estado da arte atual. São retratadas as principais fontes de umidade para a precipitação na Amazônia e o transporte de vapor d'água sobre a América do Sul. A quantificação do mecanismo de reciclagem é um indicador da importância dos processos de superfície e do clima no ciclo hidrológico, assim como da sensibilidade climática relacionada às alterações nesses processos. Os aspectos climatológicos da reciclagem na América do Sul mostram que a contribuição advectiva é mais importante para a precipitação sobre a Amazônia e o Nordeste do Brasil, ao passo que na região Centro-Sul a contribuição local tem importante papel na precipitação. Estima-se que a reciclagem de precipitação na Amazônia é da ordem de 20-35%. A advecção de umidade domina o fornecimento de vapor d'água em grande parte da região amazônica, entretanto, o papel da evapotranspiração local na reciclagem é mais importante no setor sul da bacia. Embora os estudos sobre reciclagem tenham produzido novos conhecimentos acerca da interação entre os processos de superfície e o ciclo hidrológico, os efeitos das mudanças climáticas globais nesse mecanismo ainda não estão completamente compreendidos.

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

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


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

  10. Hyper production of alkaline protease by mutagenized bacillus subtilis

    International Nuclear Information System (INIS)

    Qureshi, A.M.; Tanseem, F.


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

  11. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)



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

  12. Efficient system of artificial oil bodies for functional expression and purification of recombinant nattokinase in Escherichia coli. (United States)

    Chiang, Chung-Jen; Chen, Hong-Chen; Chao, Yun-Peng; Tzen, Jason T C


    Nattokinase, a serine protease, and pronattokinase, when expressed in Escherichia coli, formed insoluble aggregates without enzymatic activity. For functional expression and purification, nattokinase or pronattokinase was first overexpressed in E. coli as an insoluble recombinant protein linked to the C terminus of oleosin, a structural protein of seed oil bodies, by an intein fragment. Artificial oil bodies were reconstituted with triacylglycerol, phospholipid, and the insoluble recombinant protein thus formed. Soluble nattokinase was subsequently released through self-splicing of intein induced by temperature alteration, with the remaining oleosin-intein residing in oil bodies and the leading propeptide of pronattokinase, when present, spontaneously cleaved in the process. Active nattokinase with fibrinolytic activity was harvested by concentrating the supernatant. Nattokinase released from oleosin-intein-pronattokinase exhibited 5 times higher activity than that released from oleosin-intein-nattokinase, although the production yields were similar in both cases. Furthermore, active nattokinase could be harvested in the same system by fusing pronattokinase to the N terminus of oleosin via a different intein linker, with self-splicing induced by 1,4-dithiothreitol. These results have shown a great potential of this system for bacterial expression and purification of functional recombinant proteins.

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

    African Journals Online (AJOL)

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

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

    Directory of Open Access Journals (Sweden)

    Lai Meng-Jiun


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

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

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    Michael Schuliga


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

  16. Factor VII-activating protease

    DEFF Research Database (Denmark)

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


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


    Directory of Open Access Journals (Sweden)

    Iolanda Aparecida Nunes


    Full Text Available

    Its was analised 53 chicken carcasses and commercial cuts acquired in supermarkets of Goiânia-GO aiming to isolate Salmonella. The contamination rate was 13.2% and serotypes S. brandenburg, S. typhimurium, S. agona, S. derby and S. hador were identified.

    KEY-WORDS: Salmonella; chicken; chicken carcasses; commercial cuts.

    No presente estudo foram analisadas, com vistas ao isolamento de Salmonella, 53 amostras de carcaças e cortes comerciais de frangos colhidas em supermercados e casas de carnes de Goiânia - GO. Observou-se freqüência média do patógeno de 13,2%, sendo identificados os sorotipos E. brandenburg, S. typhimurium, S. agona, S. derby e S. Hadar.

    PALAVRAS-CHAVE: Salmonella; frango de corte; carcaça; cortes comerciais.

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

    African Journals Online (AJOL)

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

  19. Cysteine Protease Zymography: Brief Review. (United States)

    Wilkesman, Jeff


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

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

    NARCIS (Netherlands)

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


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

  1. La medicina Xinesa a la Xina contemporània


    Ruiz Morilla, Maria Dolors


    La medicina occidental és la imperant a tot el planeta. Malgrat tot, a la Xina la medicina tradicional hi té un pes important. La convivència entre totes dues visions de la salut i la malaltia -l'occidental i l'oriental- no és senzilla. En aquest treball es fa un recorregut pel sistema de salut xinès per a fer una aproximació a la situació de la medicina xinesa a la Xina contemporània. La medicina occidental es la imperante en todo el planeta. A pesar de todo, en China la medicina tradicio...

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


    Kyöstiö, Sirkka R. M.


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

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

    DEFF Research Database (Denmark)

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


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

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

    Directory of Open Access Journals (Sweden)

    Takayuki Shindo

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

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

    Directory of Open Access Journals (Sweden)

    Zahra Ghobadi Nejad


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

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

    Energy Technology Data Exchange (ETDEWEB)

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


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

  7. Fifteen years of HIV Protease Inhibitors: raising the barrier to resistance. (United States)

    Wensing, Annemarie M J; van Maarseveen, Noortje M; Nijhuis, Monique


    HIV protease plays a crucial role in the viral life cycle and is essential for the generation of mature infectious virus particles. Detailed knowledge of the structure of HIV protease and its substrate has led to the design of specific HIV protease inhibitors. Unfortunately, resistance to all protease inhibitors (PIs) has been observed and the genetic basis of resistance has been well documented over the past 15 years. The arrival of the early PIs was a pivotal moment in the development of antiretroviral therapy. They made possible the dual class triple combination therapy that became known as HAART. However, the clinical utility of the first generation of PIs was limited by low bioavailability and high pill burdens, which ultimately reduced adherence and limited long-term viral inhibition. When therapy failure occurred multiple protease resistance mutations were observed, often resulting in broad class resistance. To combat PI-resistance development, second-generation approaches have been developed. The first advance was to increase the level of existing PIs in the plasma by boosting with ritonavir. The second was to develop novel PIs with high potency against the known PI-resistant HIV protease variants. Both approaches increased the number of protease mutations required for clinical resistance, thereby raising the genetic barrier. This review provides an overview of the history of protease inhibitor therapy, its current status and future perspectives. It forms part of a special issue of Antiviral Research marking the 25th anniversary of antiretroviral drug discovery and development, vol. 85, issue 1, 2010. Copyright 2009 Elsevier B.V. All rights reserved.

  8. Distorted secretory granule composition in mast cells with multiple protease deficiency. (United States)

    Grujic, Mirjana; Calounova, Gabriela; Eriksson, Inger; Feyerabend, Thorsten; Rodewald, Hans-Reimer; Tchougounova, Elena; Kjellén, Lena; Pejler, Gunnar


    Mast cells are characterized by an abundance of secretory granules densely packed with inflammatory mediators such as bioactive amines, cytokines, serglycin proteoglycans with negatively charged glycosaminoglycan side chains of either heparin or chondroitin sulfate type, and large amounts of positively charged proteases. Despite the large biological impact of mast cell granules and their contents on various pathologies, the mechanisms that regulate granule composition are incompletely understood. In this study, we hypothesized that granule composition is dependent on a dynamic electrostatic interrelationship between different granule compounds. As a tool to evaluate this possibility, we generated mice in which mast cells are multideficient in a panel of positively charged proteases: the chymase mouse mast cell protease-4, the tryptase mouse mast cell protease-6, and carboxypeptidase A3. Through a posttranslational effect, mast cells from these mice additionally lack mouse mast cell protease-5 protein. Mast cells from mice deficient in individual proteases showed normal morphology. In contrast, mast cells with combined protease deficiency displayed a profound distortion of granule integrity, as seen both by conventional morphological criteria and by transmission electron microscopy. An assessment of granule content revealed that the distorted granule integrity in multiprotease-deficient mast cells was associated with a profound reduction of highly negatively charged heparin, whereas no reduction in chondroitin sulfate storage was observed. Taken together with previous findings showing that the storage of basic proteases conversely is regulated by anionic proteoglycans, these data suggest that secretory granule composition in mast cells is dependent on a dynamic interrelationship between granule compounds of opposite electrical charge.

  9. Método simples e acessível para determinar amônia liberada pela cama aviária

    Directory of Open Access Journals (Sweden)

    Hernandes Renata


    Full Text Available A amônia é um gás altamente irritante e causador de estresse às aves confinadas, o que leva à perda de peso, podendo até causar a morte. Por esse motivo, existe grande interesse em se determinar a quantidade de amônia liberada por diferentes camas aviárias e de diferentes condições, bem como os fatores que influenciam a produção desta substância. Assim, idealizou-se o presente trabalho com o objetivo de desenvolver e padronizar uma metodologia simples, acessível, exeqüível para avaliar camas de frangos submetidas a diferentes condições (diferentes tratamentos, tempos de confinamento e temperatura e capaz de determinar a potencialidade da liberação de amônia pela cama de frangos nos galpões. A metodologia baseou-se na adaptação do método da microdifusão, que consta da incubação da cama em um frasco de vidro com capacidade de 500 mL. Sobre a superfície do material incubado coloca-se um béquer de 50 mL contendo 10 mL de solução de ácido bórico 2% (m/v para fixar a amônia exalada pela cama aviária. Esse sistema é fechado e incubado por 15 horas a 30°C e, após, a solução fixadora é retirada e titulada com ácido sulfúrico 0,05 N, usando como indicador uma mistura de verde de bromocresol e alaranjado de metila. O método proposto apresenta precisão para ser usado em experimentos e utiliza material barato e reagentes facilmente encontrados nos laboratórios.

  10. Plasmodium subtilisin-like protease 1 (SUB1): insights into the active-site structure, specificity and function of a pan-malaria drug target. (United States)

    Withers-Martinez, Chrislaine; Suarez, Catherine; Fulle, Simone; Kher, Samir; Penzo, Maria; Ebejer, Jean-Paul; Koussis, Kostas; Hackett, Fiona; Jirgensons, Aigars; Finn, Paul; Blackman, Michael J


    Release of the malaria merozoite from its host erythrocyte (egress) and invasion of a fresh cell are crucial steps in the life cycle of the malaria pathogen. Subtilisin-like protease 1 (SUB1) is a parasite serine protease implicated in both processes. In the most dangerous human malarial species, Plasmodium falciparum, SUB1 has previously been shown to have several parasite-derived substrates, proteolytic cleavage of which is important both for egress and maturation of the merozoite surface to enable invasion. Here we have used molecular modelling, existing knowledge of SUB1 substrates, and recombinant expression and characterisation of additional Plasmodium SUB1 orthologues, to examine the active site architecture and substrate specificity of P. falciparum SUB1 and its orthologues from the two other major human malaria pathogens Plasmodium vivax and Plasmodium knowlesi, as well as from the rodent malaria species, Plasmodium berghei. Our results reveal a number of unusual features of the SUB1 substrate binding cleft, including a requirement to interact with both prime and non-prime side residues of the substrate recognition motif. Cleavage of conserved parasite substrates is mediated by SUB1 in all parasite species examined, and the importance of this is supported by evidence for species-specific co-evolution of protease and substrates. Two peptidyl alpha-ketoamides based on an authentic PfSUB1 substrate inhibit all SUB1 orthologues examined, with inhibitory potency enhanced by the presence of a carboxyl moiety designed to introduce prime side interactions with the protease. Our findings demonstrate that it should be possible to develop 'pan-reactive' drug-like compounds that inhibit SUB1 in all three major human malaria pathogens, enabling production of broad-spectrum antimalarial drugs targeting SUB1. Copyright © 2012 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

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

    NARCIS (Netherlands)

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


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

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

    NARCIS (Netherlands)

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

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

  13. Functional dissection of the alphavirus capsid protease: sequence requirements for activity. (United States)

    Thomas, Saijo; Rai, Jagdish; John, Lijo; Günther, Stephan; Drosten, Christian; Pützer, Brigitte M; Schaefer, Stephan


    The alphavirus capsid is multifunctional and plays a key role in the viral life cycle. The nucleocapsid domain is released by the self-cleavage activity of the serine protease domain within the capsid. All alphaviruses analyzed to date show this autocatalytic cleavage. Here we have analyzed the sequence requirements for the cleavage activity of Chikungunya virus capsid protease of genus alphavirus. Amongst alphaviruses, the C-terminal amino acid tryptophan (W261) is conserved and found to be important for the cleavage. Mutating tryptophan to alanine (W261A) completely inactivated the protease. Other amino acids near W261 were not having any effect on the activity of this protease. However, serine protease inhibitor AEBSF did not inhibit the activity. Through error-prone PCR we found that isoleucine 227 is important for the effective activity. The loss of activity was analyzed further by molecular modelling and comparison of WT and mutant structures. It was found that lysine introduced at position 227 is spatially very close to the catalytic triad and may disrupt electrostatic interactions in the catalytic site and thus inactivate the enzyme. We are also examining other sequence requirements for this protease activity. We analyzed various amino acid sequence requirements for the activity of ChikV capsid protease and found that amino acids outside the catalytic triads are important for the activity.


    Directory of Open Access Journals (Sweden)

    Alberto Augusto Eichman Jakob

    Full Text Available Resumo O objetivo principal deste artigo é analisar a migração na Amazônia brasileira. São analisados os migrantes provenientes de outros países, em especial os transfronteiriços, assim como os migrantes brasileiros. A ideia é observar também qual o impacto destes migrantes na localidade de destino na Amazônia e o papel das cidades fronteiriças neste processo. Serão estas cidades apenas de passagem para a entrada no país ou possuem atrativos suficientes para que uma parte dos migrantes fique nelas? Estariam elas prontas para receber um volume substancial de migrantes? Estes migrantes vêm diretamente de seus países ou possuem experiência prévia em outras Unidades da Federação brasileiras? Para tentar esclarecer isto, são utilizados dados do Censo Demográfico de 2010 e da Contagem Populacional de 2007 para se analisar as características dos migrantes segundo sua origem, assim como seu local escolhido de destino. A partir das análises deste trabalho, percebe-se que o perfil da migração segundo a origem é diferente, e também a escolha do local de destino, possivelmente em função das redes sociais, que têm um papel importante neste processo de atração de migrantes em determinadas áreas.

  15. Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae. (United States)

    Wilkinson, Derek; Ramsdale, Mark


    A variety of proteases have been implicated in yeast PCD (programmed cell death) including the metacaspase Mca1 and the separase Esp1, the HtrA-like serine protease Nma111, the cathepsin-like serine carboxypeptideases and a range of vacuolar proteases. Proteasomal activity is also shown to have an important role in determining cell fate, with both pro- and anti-apoptotic roles. Caspase 3-, 6- and 8-like activities are detected upon stimulation of yeast PCD, but not all of this activity is associated with Mca1, implicating other proteases with caspase-like activity in the yeast cell death response. Global proteolytic events that accompany PCD are discussed alongside a consideration of the conservation of the death-related degradome (both at the level of substrate choice and cleavage site). The importance of both gain-of-function changes in the degradome as well as loss-of-function changes are highlighted. Better understanding of both death-related proteases and their substrates may facilitate the design of future antifungal drugs or the manipulation of industrial yeasts for commercial exploitation.

  16. Distinct pathways of mannan-binding lectin (MBL)- and C1-complex autoactivation revealed by reconstitution of MBL with recombinant MBL-associated serine protease-2

    DEFF Research Database (Denmark)

    Vorup-Jensen, T; Petersen, Steen Vang; Hansen, A G


    proteolytic activation of both C1r and C1s, reconstitution with MASP-2 alone is sufficient for complement activation by MBL. The results suggest that the catalytic activities of MASP-2 split between the two proteases of the C1 complex during the course of vertebrate complement evolution. Udgivelsesdato: 2000...

  17. Adaptabilidade e estabilidade de cultivares de soja em cinco épocas de plantio no cerrado de Rondônia

    Directory of Open Access Journals (Sweden)

    Prado Eloi Elias do


    Full Text Available Este trabalho teve o objetivo de avaliar o desempenho, a estabilidade e a adaptabilidade de 21 cultivares de soja (Glycine max (L. Merrill, em cinco épocas de plantio no cerrado de Rondônia. O delineamento experimental foi blocos ao acaso, com três repetições. Os ensaios foram conduzidos no ano de 1996/97 pelo Centro de Pesquisa Agroflorestal de Rondônia, no Campo Experimental de Vilhena em Rondônia, RO. Os métodos utilizados apresentaram concordância entre os resultados alcançados, sendo que o comportamento das cultivares nas diferentes épocas de semeadura pode ser representado por um modelo linear. O coeficiente de resposta linear beta1i não apresentou diferença significativa (P<0,05 da unidade pelo teste t (beta1i = 1 em todas as cultivares testadas. As cultivares que apresentaram desvios de regressão sigma²(d i significativos (P<0,05 foram MT/BR50 e EMBRAPA 31, com R² = 58,56% e 74,43%, respectivamente. As demais cultivares não apresentaram desvios de regressão significativos.

  18. Economia na produção de helicônias no Estado do Rio de Janeiro Economy in helicônias production in Rio de Janeiro State

    Directory of Open Access Journals (Sweden)

    Andrezza da Silva Machado Neto


    Full Text Available A pesquisa teve como objetivo determinar, mediante o cálculo do valor presente líquido (VPL, da taxa interna de retorno (TIR e do Payback Econômico (PBE, a viabilidade da produção de helicônias no Estado do Rio de Janeiro, bem como identificar e verificar, por meio da análise de sensibilidade, em que medida alguns fatores do sistema produtivo exercem impacto sobre a rentabilidade das regiões produtoras. Os resultados mostraram que a produção de helicônias é uma atividade lucrativa, segundo o método da TIR e considerando taxas mínimas de atratividade (TMA de 6% e 12% para o cálculo do VPL. Porém, ocorrem limitações no setor de escoamento da produção, retardando a consolidação desse mercado. O percentual de perdas e o preço de venda do produto apresentaram-se como fatores determinantes sobre o tempo de retorno do investimento (PBE. Além disso, o preço de venda do produto foi a variável com maior efeito sobre a rentabilidade, seguida do valor da terra e do custo das mudas.The purpose of the research was to determine, through the calculation of net present value (VPL, internal return rate (TIR and Economic Payback (PBE, the viability of Heliconias production in the Rio de Janeiro State, as well as identify and verify, by means of sensitivity analysis in which extent some factors of the production system exercise impact on the profitability of the producing regions. The results showed that helicônia production is a profitable activity, according to the method of TIR and considering minimal attractiveness rates (TMA of 6% and 12% for calculating the net present value (VPL. However, there are limitations in the sector of produce flow, delaying the consolidation of this market. The percentage of losses and the selling price of the product presented determinant factors on the time of return on investment (PBE. In addition, the selling price of the product was the variable with greater effect on profitability, followed by the

  19. The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying. (United States)

    Wang, Shuaiyu; Jacquemyn, Julie; Murru, Sara; Martinelli, Paola; Barth, Esther; Langer, Thomas; Niessen, Carien M; Rugarli, Elena I


    The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both Afg3l2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.

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

    African Journals Online (AJOL)

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

  1. A sexualidade para o adolescente em situação de rua em Goiânia La sexualidad para el adolescente callejero de Goiânia The meaning of sexuality for adolescents living on the street in Goiânia

    Directory of Open Access Journals (Sweden)

    Marcelo Medeiros


    Full Text Available Este estudo buscou uma reflexão sobre a sexualidade do adolescente em situação de rua partindo da ótica destes. Pesquisa de abordagem qualitativa objetivando identificar aspectos gerais sobre a sexualidade de jovens que vivem nas ruas na cidade de Goiânia. Os dados foram coletados através de entrevista semi-estruturada e observação participante. Os resultados mostraram que para os meninos e meninas a sexualidade está reduzida ao ato sexual e que não se privam deste na rua sendo, a prevenção uma atitude praticamente inexistente no seu cotidiano. Apontamos para a importância do enfermeiro comprometer-se com esse grupo através de trabalhos de educação e promoção da saúde.Este estudio buscó una reflexión sobre la sexualidad de los adolescentes que viven en la calle. Investigación de abordaje cualitativo con el objetivo de identificar los aspectos generales sobre la sexualidad de los jóvenes que viven en las calles de la ciudad de Goiânia. Los datos fueron obtenidos por medio de una entrevista semi-estructurada y por observación participante. Los resultados mostraron que tanto para los hombres como para las mujeres, la sexualidad se reduce al acto sexual y que ellos no se privan de este en la calle; la prevención es una actitud prácticamente inexistente en el cotidiano de ellos. Así, nosotros señalamos lo importante que es el hecho del enfermero comprometerse con ese grupo realizando actividades de educación y promoción de la salud.This study aimed at reflecting on the sexuality for adolescents living on the street, based on their point of view. Authors used a qualitative approach in order to identify general aspects related to the sexuality of adolescents living on the street in Goiânia. Data were collected through semi-structured interviews and participant observation. Results showed that these adolescents consider the sexuality reduced to the sexual act, and that even on the streets they are not deprived of it. Also

  2. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease. (United States)

    Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F


    FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

  3. Núcleos coloniais e agricultura na Amazônia Imperial: uso e ocupação da terra

    Directory of Open Access Journals (Sweden)

    Francivaldo Alves Nunes


    Full Text Available Fomento a produção, reforma das técnicas agrícolas, intensificação dos sistemas produtivos e aumento da produtividade são algumas diretrizes recorrentes nos projetos de reformulação da agricultura desenvolvidos no Brasil no período Imperial. Na Amazônia a criação dos Núcleos Coloniais, na segunda metade do século XIX, constituem ações governamentais que buscam atender essas demandas. Analisar os elementos que estimulam a formação das Colônias Agrícolas e que nos ajuda a pensar a constituição de paisagens agrárias e os processos de apropriação da terra é a tarefa que nos ocupamos nesta comunicação; entendendo que, além de uma questão de consumo e produção, os Núcleos Coloniais na Amazônia se evidenciaram como espaços de experimentação de novas técnicas produtivas, melhor aproveitamento da terra, do exercício de domínio das matas, promoção do povoamento e disciplinarização dos sujeitos sociais envolvidos na construção deste espaço.

  4. Studies on detection and analysis of proteases in leaf extract of medicinally important plants. (United States)

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


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

  5. Protease activation involved in resistance of human cells to x-ray cell killing

    International Nuclear Information System (INIS)

    Zhang, Hong-Chang; Takahashi, Shuji; Karata, Kiyonobu; Kita, Kazuko; Suzuki, Nobuo


    Little is known of proteases that play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa-R cells after X-ray irradiation. The activity was identified as fibrinolytic, using 125 I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa-R cells showed an increased level of protease activity 10 min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with the X-ray susceptibility of cells. Leupeptin, a serine-cysteine protease inhibitor, inhibited the protease activity in samples obtained from X-ray-irradiated RSa-R cells. Treatment of RSa-R cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. However, the treatment of cells with other inhibitors tested did not modulate the X-ray susceptibility. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. (author)

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

    LENUS (Irish Health Repository)

    Greene, Catherine M


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

  7. Invention et interprétation : chants de boisson et chants chamaniques chez les Suruí du Rondônia


    Yvinec, Cédric


    Invention et interprétation : chants de boisson et chants chamaniques chez les Suruí du Rondônia. Cet article examine les techniques rhétoriques mises en œuvre par deux genres de chants rituels des Suruí du Rondônia, chants de fête de boisson et chants chamaniques, notamment les écarts par lesquels la langue et l’énonciation rituelles se distinguent du discours ordinaire. Ces procédés rhétoriques trouvent un écho au niveau sociologique, à travers le statut du chamane et celui du grand homme d...

  8. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    International Nuclear Information System (INIS)

    Jin Xin; Li Jufang; Huang Pingying; Dong Xuyan; Guo Lulu; Yang Liang; Cao Yuancheng; Wei Fang; Zhao Yuandi


    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  9. Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

    Energy Technology Data Exchange (ETDEWEB)

    Jin Xin [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Li Jufang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Huang Pingying [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Dong Xuyan [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Guo Lulu [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Yang Liang; Cao Yuancheng [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China); Wei Fang [Key Lab of Oil Crops Biology, Ministry of Agriculture, Institute of Oil Crops Research, Chinese Academy of Agricultural Sciences, Wuhan, Hubei 430062 (China); Zhao Yuandi, E-mail: [Wuhan National Laboratory for Optoelectronics-Hubei Bioinformatics and Molecular Imaging Key Laboratory, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan, HuBei 430074 (China)


    (3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63+-2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

  10. Single-Step Purification and Characterization of A Recombinant Serine Proteinase Inhibitor from Transgenic Plants. (United States)

    Jha, Shweta; Agarwal, Saurabh; Sanyal, Indraneel; Amla, D V


    Expression of recombinant therapeutic proteins in transgenic plants has a tremendous impact on safe and economical production of biomolecules for biopharmaceutical industry. The major limitation in their production is downstream processing of recombinant protein to obtain higher yield and purity of the final product. In this study, a simple and rapid process has been developed for purification of therapeutic recombinant α1-proteinase inhibitor (rα1-PI) from transgenic tomato plants, which is an abundant serine protease inhibitor in human serum and chiefly inhibits the activity of neutrophil elastase in lungs. We have expressed rα1-PI with modified synthetic gene in transgenic tomato plants at a very high level (≃3.2 % of total soluble protein). The heterologous protein was extracted with (NH4)2SO4 precipitation, followed by chromatographic separation on different matrices. However, only immunoaffinity chromatography resulted into homogenous preparation of rα1-PI with 54 % recovery. The plant-purified rα1-PI showed molecular mass and structural conformation comparable to native serum α1-PI, as shown by mass spectrometry and optical spectroscopy. The results of elastase inhibition assay revealed biological activity of the purified rα1-PI protein. This work demonstrates a simple and efficient one-step purification of rα1-PI from transgenic plants, which is an essential prerequisite for further therapeutic development.

  11. Maximized Autotransporter-Mediated Expression (MATE for Surface Display and Secretion of Recombinant Proteins in Escherichia coli

    Directory of Open Access Journals (Sweden)

    Shanna Sichwart


    Full Text Available A new optimized system for the surface display and secretion of recombinant proteins is described, termed MATE (maximized autotransporter-mediated expression. It is based on an artificial gene consisting of the coding region for the signal peptide of CtxB, a multiple cloning site for passenger gene insertion, flanked by coding sequences for linear epitopes for monoclonal antibodies and OmpT, and factor Xa protease cleavage sites followed by a codon-optimized DNA sequence of the linker and the β-barrel of the type V autotransporter EhaA from Escherichia coli under control of an IPTG-inducible T5 promoter. The MATE system enabled the continuous secretion of recombinant passenger mCherry via OmpT-mediated cleavage, using native OmpT protease activity in E. coli when grown at 37 °C. It is the first example to show that native OmpT activity is sufficient to facilitate the secretion of a correctly folded target protein in preparative amounts obtaining 240 μg of purified mCherry from 800 mL of crude culture supernatant. Because the release of mCherry was achieved by a simple transfer of the encoding plasmid from an OmpT-negative to an OmpT-positive strain, it bears the option to use surface display for screening purposes and secretion for production of the selected variant. A single plasmid could therefore be used for continuous secretion in OmpT-positive strains or surface display in OmpT-negative strains. In conclusion, the MATE system appears to be a versatile tool for the surface display and for the secretion of target proteins in E. coli.

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

    DEFF Research Database (Denmark)

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


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

  13. Characterization of DegQVh, a serine protease and a protective immunogen from a pathogenic Vibrio harveyi strain. (United States)

    Zhang, Wei-wei; Sun, Kun; Cheng, Shuang; Sun, Li


    Vibrio harveyi is an important marine pathogen that can infect a number of aquaculture species. V. harveyi degQ (degQ(Vh)), the gene encoding a DegQ homologue, was cloned from T4, a pathogenic V. harveyi strain isolated from diseased fish. DegQ(Vh) was closely related to the HtrA family members identified in other Vibrio species and could complement the temperature-sensitive phenotype of an Escherichia coli strain defective in degP. Expression of degQ(Vh) in T4 was modulated by temperature, possibly through the sigma(E)-like factor. Enzymatic analyses demonstrated that the recombinant DegQ(Vh) protein expressed in and purified from E. coli was an active serine protease whose activity required the integrity of the catalytic site and the PDZ domains. The optimal temperature and pH of the recombinant DegQ(Vh) protein were 50 degrees C and pH 8.0. A vaccination study indicated that the purified recombinant DegQ(Vh) was a protective immunogen that could confer protection upon fish against infection by V. harveyi. In order to improve the efficiency of DegQ(Vh) as a vaccine, a genetic construct in the form of the plasmid pAQ1 was built, in which the DNA encoding the processed DegQ(Vh) protein was fused with the DNA encoding the secretion region of AgaV, an extracellular beta-agarase. The E. coli strain harboring pAQ1 could express and secrete the chimeric DegQ(Vh) protein into the culture supernatant. Vaccination of fish with viable E. coli expressing chimeric degQ(Vh) significantly (P < 0.001) enhanced the survival of fish against V. harveyi challenge, which was possibly due to the relatively prolonged exposure of the immune system to the recombinant antigen produced constitutively, albeit at a gradually decreasing level, by the carrier strain.

  14. Ocorrência de Herminodes sp. (Lepidoptera: Noctuidae em pupunheira nos estados do Acre e Rondônia, Brasil Occurrence of Herminodes sp. (Lepidoptera: Noctuidae in peach palm in the States of Acre and Rondônia, Brazil

    Directory of Open Access Journals (Sweden)

    Marcílio José Thomazini


    Full Text Available É relatado a ocorrência de Herminodes sp. em plantas de pupunha na área experimental da Embrapa Acre, Rio Branco, AC e no Projeto Reca, Vila Nova Califórnia, RO, Brasil. São descritas as principais características deste inseto e os danos por ele causados. Este é o primeiro registro de lagartas do gênero Herminodes causando dano em pupunheiras nos estados do Acre e Rondônia.Related herein is the occurrence of Herminodes sp. in peach palm plants at experimental area of Embrapa Acre, Rio Branco, AC and at Projeto Reca, Vila Nova California, RO, Brazil. The main characteristics of this insect and the damage it causes are described. This is the first record of Herminodes caterpillars damaging peach palm in the states of Acre and Rondônia.

  15. Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

    Energy Technology Data Exchange (ETDEWEB)

    Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin


    The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

  16. Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum. (United States)

    Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li


    Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  17. Recombinant Programming


    Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman


    This research report presents a promising new approach to computation called Recombinant Programming. The novelty of our approach is that it separates the program into two layers of computation: the recombination and the interpretation layer. The recombination layer takes sequences as inputs and allows the programmer to recombine these sequences through the definition of cohesive code units called extensions. The output of such recombination is a mesh that can be used by the interpretation la...

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


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


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

  19. Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus. (United States)

    Cuenca-Soria, C A; Álvarez-González, C A; Ortiz-Galindo, J L; Nolasco-Soria, H; Tovar-Ramírez, D; Guerrero-Zárate, R; Castillo-Domínguez, A; Perera-García, M A; Hernández-Gómez, R; Gisbert, E


    The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

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

    African Journals Online (AJOL)



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

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

    African Journals Online (AJOL)



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

  2. Modelo de desenvolvimento local na Amazônia Pré-Colonial: complexidade cultural e modernidade em sociedades pré-coloniais da Amazônia

    Directory of Open Access Journals (Sweden)

    Edinaldo Pinheiro Nunes Filho


    Full Text Available Normal 0 21 false false false MicrosoftInternetExplorer4 Este artigo retrata a cultura material das sociedades complexas pré-coloniais que existiram nos estados do Pará e Amapá e destaca a importância do trabalho interdisciplinar como meio para que as evidências arqueológicas colaborem a se repensar a relação entre as populações humanas e o meio ambiente na Amazônia pré-colonial.  Assim, o artigo trabalha a complexidade cultural como conseqüência de desenvolvimento sustentável.

  3. Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria. (United States)

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


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



    Noll, Matias; Candotti, Cláudia Tarragô; Rosa, Bruna Nichele da; Schoenell, Maira Cristina Wolf; Tiggemann, Carlos Leandro; Loss, Jefferson Fagundes


    to investigate the prevalence of back pain during a prior three-month period; to identify postural and behavioral habits; to assess whether a relationship exists between back pain and the postural and behavioral habits of elementary school students in the municipal school network in the city of Teutônia, Rio Grande do Sul (RS). METHODS: this was an epidemiological population study in which 833 5th to 8th grade students from schools in the municipal school network in Teutônia participated. The...

  5. Retratos próprios e desconhecidos: experiências de fronteiras (? E diálogos artísticos nas Amazônias Ocidental e Oriental

    Directory of Open Access Journals (Sweden)

    John Fletcher


    Full Text Available O presente trabalho apresenta uma interpretação crí- tica e social de dois projetos artísticos apresentados na cidade de Popayán, Colômbia, localizada na Amazônia Ocidental, em diálogo com outro projeto visual realizado na cidade de Belém, Pará, Brasil, território pertencente à Amazônia Oriental. Em termos conceituais, o mesmo será amparado por alguns eixos de pensamentos críticos como os de Clifford Geertz, Raymond Williams, Pierre Bordieu, Chantal Mouffe, Ernesto Laclau, Mikhail Bakhtin e Nestor García Canclini, bem como buscará destacar uma metodologia polifônica, em muito articulada com alguns dos pressupostos da participação de distintos sujeitos para um agenciamento liminar do pensamento Latino Americano. É sob estes marcadores contextuais, portanto, que a pesquisa se alia a uma operação interpretativa para se rascunhar e debater aproximações e distanciamentos alocados no interior destas Amazônias, cujas visibilidades dão sustentação para horizontes dinâmicos, talvez mais inclusivos e menos autoritários. Palavras-chave: Arte Contemporânea, Interpretação, Amazônia, Colômbia.

  6. Identification and characterization of fusolisin, the Fusobacterium nucleatum autotransporter serine protease.

    Directory of Open Access Journals (Sweden)

    Lior Doron

    Full Text Available Fusobacterium nucleatum is an oral anaerobe associated with periodontal disease, adverse pregnancy outcomes and colorectal carcinoma. A serine endopeptidase of 61-65 kDa capable of damaging host tissue and of inactivating immune effectors was detected previously in F. nucleatum. Here we describe the identification of this serine protease, named fusolisin, in three oral F. nucleatum sub-species. Gel zymogram revealed fusobacterial proteolytic activity with molecular masses ranging from 55-101 kDa. All of the detected proteases were inhibited by the serine protease inhibitor PMSF. analysis revealed that all of the detected proteases are encoded by genes encoding an open reading frame (ORF with a calculated mass of approximately 115 kDa. Bioinformatics analysis of the identified ORFs demonstrated that they consist of three domains characteristic of autotransporters of the type Va secretion system. Our results suggest that the F. nucleatum fusolisins are derived from a precursor of approximately 115 kDa. After crossing the cytoplasmic membrane and cleavage of the leader sequence, the C-terminal autotransporter domain of the remaining 96-113 kDa protein is embedded in the outer membrane and delivers the N-terminal S8 serine protease passenger domain to the outer cell surface. In most strains the N-terminal catalytic 55-65 kDa domain self cleaves and liberates itself from the autotransporter domain after its transfer across the outer cell membrane. In F. nucleatum ATCC 25586 this autocatalytic activity is less efficient resulting in a full length membrane-anchored serine protease. The mature serine protease was found to cleave after Thr, Gly, Ala and Leu residues at the P1 position. Growth of F. nucleatum in complex medium was inhibited when serine protease inhibitors were used. Additional experiments are needed to determine whether fusolisin might be used as a target for controlling fusobacterial infections.

  7. Uncoupling of Protease trans-Cleavage and Helicase Activities in Pestivirus NS3. (United States)

    Zheng, Fengwei; Lu, Guoliang; Li, Ling; Gong, Peng; Pan, Zishu


    The nonstructural protein NS3 from the Flaviviridae family is a multifunctional protein that contains an N-terminal protease and a C-terminal helicase, playing essential roles in viral polyprotein processing and genome replication. Here we report a full-length crystal structure of the classical swine fever virus (CSFV) NS3 in complex with its NS4A protease cofactor segment (PCS) at a 2.35-Å resolution. The structure reveals a previously unidentified ∼2,200-Å 2 intramolecular protease-helicase interface comprising three clusters of interactions, representing a "closed" global conformation related to the NS3-NS4A cis -cleavage event. Although this conformation is incompatible with protease trans -cleavage, it appears to be functionally important and beneficial to the helicase activity, as the mutations designed to perturb this conformation impaired both the helicase activities in vitro and virus production in vivo Our work reveals important features of protease-helicase coordination in pestivirus NS3 and provides a key basis for how different conformational states may explicitly contribute to certain functions of this natural protease-helicase fusion protein. IMPORTANCE Many RNA viruses encode helicases to aid their RNA genome replication and transcription by unwinding structured RNA. Being naturally fused to a protease participating in viral polyprotein processing, the NS3 helicases encoded by the Flaviviridae family viruses are unique. Therefore, how these two enzyme modules coordinate in a single polypeptide is of particular interest. Here we report a previously unidentified conformation of pestivirus NS3 in complex with its NS4A protease cofactor segment (PCS). This conformational state is related to the protease cis -cleavage event and is optimal for the function of helicase. This work provides an important basis to understand how different enzymatic activities of NS3 may be achieved by the coordination between the protease and helicase through different

  8. Biochemical characterization of a halophilic, alkalithermophilic protease from Alkalibacillus sp. NM-Da2. (United States)

    Abdel-Hamed, Asmaa R; Abo-Elmatty, Dina M; Wiegel, Juergen; Mesbah, Noha M


    An extracellular, halophilic, alkalithermophilic serine protease from the halo-alkaliphilic Alkalibacillus sp. NM-Da2 was purified to homogeneity by ethanol precipitation and anion-exchange chromatography. The purified protease was a monomeric enzyme with an approximate molecular mass of 35 kDa and exhibited maximal activity at 2.7 M NaCl, pH 55 °C 9 and 56 °C. The protease showed great temperature stability, retaining greater than 80 % of initial activity after 2 h incubation at 55 °C. The protease was also extremely pH tolerant, retaining 80 % of initial activity at pH 55 °C 10.5 after 30 min incubation. Protease hydrolyzed complex substrates, displaying activity on yeast extract, tryptone, casein, gelatin and peptone. Protease activity was inhibited at casein concentrations greater than 1.2 mg/mL. The enzyme was stable and active in 40 % (v/v) solutions of isopropanol, ethanol and benzene and was stable in the presence of the polysorbate surfactant Tween 80. Activity was stimulated with the oxidizing agent hydrogen peroxide. Inhibition with phenyl methylsulfonylfluoride indicates it is a serine protease. Synthetic saline wastewater treated with the protease showed 50 % protein removal after 5 h. Being halophilic, alkaliphilic and thermophilic, in addition to being resistant to organic solvents, this protease has potential for various applications in biotechnological and pharmaceutical industries.

  9. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.


    Purpose: To shorten the pre-heating time for a recombiner and obtain a uniform temperature distribution for the charged catalyst layer in a BWR type reactor. Constitution: A pre-heating heater is disposed to the outer periphery of a vessel for a recombiner packed with catalysts for recombining hydrogen and oxygen in gases flowing through a radioactive gaseous wastes processing system. Heat pipes for transmitting the heat applied to said container to the catalyst are disposed vertically and horizontally within the container. Different length of the heat pipes are combined. In this way, pre-heating time for the recombiner before the operation start and before the system switching can be shortened and the uniform pre-heating for the inside of the recombiner is also made possible. Further, heater control in the pre-heating can be carried out effectively and with ease. (Moriyama, K.)


    Directory of Open Access Journals (Sweden)


    Full Text Available ABSTRACT In the present paper we investigated the insect gall distribution along savanna and forest sites in the Floresta Nacional de Silvânia, Goiás, Brazil. The insect gall fauna was surveyed bi-monthly between December 2009 and June 2010. In total we found 186 insect gall morphotypes, distributed on 35 botanical families and 61 plant species. Ninety-nine insect gall morphotypes were recorded in the forest and 87 in the savanna. Gall-inducing insects belonged to Coleoptera, Diptera, Hemiptera, Lepidoptera and Thysanoptera, with highlight to Cecidomyiidae (Diptera that induced 34.1% of the gall morphotypes. Parasitoids and/or inquilines were recorded in 38 morphotypes, mainly from the families Eulophidae, Eurytomidae and Torymidae (Hymenoptera. Fabaceae was the botanical family with the greatest richness of galls, followed by Asteraceae and Sapindaceae, being Protium (Burseraceae, Siparuna (Siparunaceae and Serjania (Sapindaceae the main host genera. This is the first systematic survey of insect galls realized in the Flona-Silvânia, which result in six plant species are recorded for the first time in Brazil as host of insect galls.


    Directory of Open Access Journals (Sweden)

    Rodrigo Ramos Hospodar Felippe Valverde


    Full Text Available This article intends to evaluate the spatial consequences of the ambitious cultural policy developed by the city of Paulínia (SP in 2006 and the dilemmas of its growth after 2012. More precisely, I chose to analyze the challenges of creating a film industry in Paulínia. In order to do so, I used the concept of cultural industry as seen in Scott & Power (2004, which could be synthesize as a strategy promoted by public and private agents to create at the same time an economy capable of carrying culture values as much as a culture that gains economic value. A cultural industry could promote an economic ativity capable of benefit itself from social life and use of unending resources. Besides, a cultural industry counts on fast growing consumer markets. As a result, I hope to contribute to the Brazilian Cultural Geography as I present a discussion that promote the convergence of arguments found in economics and cultural theories, in opposition to what Geography traditionally did.


    Directory of Open Access Journals (Sweden)

    Ana Maria Guimarães da Mota


    Full Text Available O presente artigo é resultado parcial das investigações doprojeto “Turismo e Desenvolvimento Socioespacial” queestá sendo desenvolvido desde abril de 2006 no CentroFederal de Educação Tecnológica de Goiás – CEFET/GO.As discussões ora apresentadas visam compreender adinâmica da Feira Hippie de Goiânia e o seu impacto geradono setor turístico-hoteleiro da cidade de Goiânia. Nosúltimos anos, esse empreendimento passou por grandestransformações de ordem social, econômica e espacialque resultaram em alterações nos equipamentos turísticos(sistema de hospedagem e restaurantes, de transportese agências de viagens e nos produtos oferecidos. Apertinência desse estudo se dá pelo papel que esta feirarepresenta na configuração do chamado “turismo de compras”.Essa problemática serviu de suporte para uma abordagemque resultou na construção de um banco de dadose informações que ajudam na compreensão da dinâmicade parte dos processos turísticos do Estado de Goiás.

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

    NARCIS (Netherlands)

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


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

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

    Directory of Open Access Journals (Sweden)

    Jiangning Song

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

  15. The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria. (United States)

    König, Tim; Tröder, Simon E; Bakka, Kavya; Korwitz, Anne; Richter-Dennerlein, Ricarda; Lampe, Philipp A; Patron, Maria; Mühlmeister, Mareike; Guerrero-Castillo, Sergio; Brandt, Ulrich; Decker, Thorsten; Lauria, Ines; Paggio, Angela; Rizzuto, Rosario; Rugarli, Elena I; De Stefani, Diego; Langer, Thomas


    Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca 2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca 2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca 2+ homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.


    Directory of Open Access Journals (Sweden)

    João Carlos Gióia


    Full Text Available

    Thirty-nine samples of honey produced in Goiânia-GO and vicinities were analysed in relation to solid solubles, moisture, acidity in formic acid reducing sugars on glucose. The search for falsification was based on LUND’s reaction, the FIEHE test, Lugol test and the determination of the diastasics ferments. Only 30.77% of the samples analyzed were within the established by the legislation.

    Foram analisadas 39 amostras de mel de abelha produzido na região de Goiânia – GO, com o objetivo de se avaliar sua qualidade. Determinou-se: sólidos solúveis, umidade, acidez em ácido fórmico e glicídios redutores em glicose. As pesquisas de fraudes basearam-se nas provas de FIEHE, de LUND, teste do lugol e determinação de fermentos diastásicos. Somente 30,77% das amostras analisadas apresentaram-se conforme os padrões estabelecidos pela legislação vigente em todas as provas realizadas.

  17. Purification and characterization of alkaline proteases from aspergillus terreus

    International Nuclear Information System (INIS)

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


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

  18. Molecular Evolution of a Type 1 Wild-Vaccine Poliovirus Recombinant during Widespread Circulation in China (United States)

    Liu, Hong-Mei; Zheng, Du-Ping; Zhang, Li-Bi; Oberste, M. Steven; Pallansch, Mark A.; Kew, Olen M.


    Type 1 wild-vaccine recombinant polioviruses were isolated from poliomyelitis patients in China from 1991 to 1993. We compared the sequences of 34 recombinant isolates over the 1,353-nucleotide (nt) genomic interval (nt 2480 to 3832) encoding the major capsid protein, VP1, and the protease, 2A. All recombinants had a 367-nt block of sequence (nt 3271 to 3637) derived from the Sabin 1 oral poliovirus vaccine strain spanning the 3′-terminal sequences of VP1 (115 nt) and the 5′ half of 2A (252 nt). The remaining VP1 sequences were closely (up to 99.5%) related to those of a major genotype of wild type 1 poliovirus endemic to China up to 1994. In contrast, the non-vaccine-derived sequences at the 3′ half of 2A were more distantly related (polioviruses from China. The vaccine-derived sequences of the earliest (April 1991) isolates completely matched those of Sabin 1. Later isolates diverged from the early isolates primarily by accumulation of synonymous base substitutions (at a rate of ∼3.7 × 10−2 substitutions per synonymous site per year) over the entire VP1-2A interval. Distinct evolutionary lineages were found in different Chinese provinces. From the combined epidemiologic and evolutionary analyses, we propose that the recombinant virus arose during mixed infection of a single individual in northern China in early 1991 and that its progeny spread by multiple independent chains of transmission into some of the most populous areas of China within a year of the initiating infection. PMID:11070012

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

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    Celia A. Schiffer


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

  20. Interdependence of Inhibitor Recognition in HIV-1 Protease. (United States)

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


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

  1. Estratégias para evitar a perda de biodiversidade na Amazônia

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    Ima Célia Guimarães Vieira


    Full Text Available A SOCIEDADE brasileira recebe, anualmente, a estimativa de perda de floresta na Amazônia por meio da taxa de desflorestamento divulgada pelo Inpe, a qual, em 2004, foi de aproximadamente 26.130 km² . O que não se conhece é o quanto de recursos naturais se perde a cada quilômetro quadrado de floresta destruída. Neste trabalho, apresentamos números concretos desta perda, baseados em estudos recentes sobre a densidade de plantas e de alguns grupos de animais na Amazônia. Com base nisso, defendemos a idéia de que não há necessidade de se ampliar o desflorestamento na região e que, portanto, qualquer licença de desmatamento deveria ser proibida na Amazônia. Sugerimos também que o sistema de ciência e tecnologia regional deve ser descentralizado através do desenvolvimento de programas de pesquisa integrados, focalizados no desenho e no teste de modelos de territórios sustentáveis para os diferentes setores da região.BRAZILIAN society receives yearly an estimate of the Amazonian rainforest loss by means of a deforestation rate announced by Inpe, which in 2004 was of approximatly 26,130 km². What we do not know is how much of the natural resources is lost in each square kilometer of forest destruction. In this work, we present concrete numbers of forest loss, based on recent studies of plant densities and some animal species in the Amazon region. Our findings support the idea that it is not necessary to extend further deforestation in the region and, therefore, that deforestation licenses should be forbidden. Moreover, we propose that regional science and technology system must be gradually descentralized and enhanced through the integration and development of research programs focused in drawing and testing models of sustainable territories for the different social segments of the region.

  2. As redes para além dos rios: urbanização e desequilíbrios na Amazônia brasileira


    Sathler, Douglas; Monte-Mór, Roberto L.; Carvalho, José Alberto Magno de


    Nas últimas décadas, as redes urbanas que se estendem sobre a Amazônia Legal têm evoluído com o surgimento de cidades médias e com a multiplicação de pequenas aglomerações urbanas, que seguem os traçados das principais rodovias e rios da região. Apesar disso, as redes da Amazônia Legal apresentam diversos aspectos que devem ser estudados sem "euforia", diante de uma série de peculiaridades que merecem ser exploradas com maior profundidade. Mesmo que tenha sido estruturada uma hierarquia urban...

  3. In vitro protein digestibility of enzymatically pre-treated bean (Phaseolus vulgaris L. flour using commercial protease and Bacillus sp. protease Digestibilidade protéica in vitro de farinhas de feijão (Phaseolus vulgaris L. pré-tratadas com protease comercial e protease de Bacillus sp.

    Directory of Open Access Journals (Sweden)

    Disney Ribeiro Dias


    Full Text Available The common bean (Phaseolus vulgaris L. is a staple food in the Brazilian diet and represents the major source of dietary protein and other micronutrients and minerals. Despite the considerable protein concentration in beans, the food is considered of low biological value when compared to animal proteins and other plant protein sources. To improve the availability of protein in beans, enzymatic treatments were performed in four cultivars (ON, OPNS, TAL and VC3. The approach was a completely randomized design with four replicates. We used a 4 × 3 factorial arrangement (four cultivars and three treatments: treatment 1-addition of commercial protease (Trypsin 250, Difco, treatment 2-addition of protease from Bacillus sp., and treatment 3:-control without enzyme addition. The enzyme: substrate ratio was 5% w/w (amount of enzyme per total protein in bean flour. The approach was a completely randomized design with four replicates. A 4 × 3 factorial arrangement (four cultivars and three treatments, the same as those mentioned above was used. The concentration of total protein (g.100 g-1 of dry matter in the samples ranged from 16.94 to 18.06%, while the concentration of total phenolics was between 0.78 and 1.12% (g Eq. tannic acid.100 g-1 dry matter. The in vitro protein digestibility of enzymatically untreated bean flour (control ranged from 47.30 to 56.17% based on the digestibility of casein. Concentrations of P, K, Ca, Mg, and Zn observed in the four cultivars tested were within the average values available in the literature. Treatment 2 with protease from Bacillus sp. induced decreases in the levels of Cu and Mn. The average Fe content increased in all bean flour samples when treated with proteases, reaching a maximum increase of 102% in the TAL flour treated with protease from Bacillus sp. The digestibility of all beans tested was significantly increased (p O feijão (Phaseolus vulgaris L. é um alimento básico na refeição do brasileiro

  4. Volatilização de Amônia Proveniente de Ureia Compactada com Enxofre e Bentonita, em Ambiente Controlado

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    José Antônio de Oliveira


    Full Text Available Nos últimos anos, novas tecnologias têm sido desenvolvidas para reduzir as perdas de N quando se utiliza ureia como fonte desse macronutriente. A utilização de fertilizantes de características ácidas pode reduzir as perdas de amônia por volatilização, quando combinados com a ureia. O objetivo deste trabalho foi avaliar as perdas de amônia provenientes de fontes de N revestidas e, ou, incorporadas com ou sem enxofre e bentonita. Esses foram aplicados na superfície de um Planossolo Háplico contido em bandejas (0,1 m2 de área e 10 cm de profundidade, em dose equivalente a 200 kg ha-1 de N. Foram avaliadas as perdas de N-NH3 por volatilização durante 21 dias, com auxílio de um coletor semiaberto. A adição de diferentes fontes de enxofre e de bentonita no processo de compactação da ureia reduziu as perdas de amônia em até 29 %, quando comparadas com a ureia granulada comercial, comprovando serem alternativas promissoras para aumentar a eficiência da adubação nitrogenada.

  5. Variable context Markov chains for HIV protease cleavage site prediction. (United States)

    Oğul, Hasan


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

  6. O Núcleo de Apoio à Saúde da Família em Goiânia (GO: percepções dos profissionais e gestores

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    Jéssica Félix Nicácio Martinez

    Full Text Available RESUMO O Núcleo de Apoio à Saúde da Família (Nasf foi criado em 2008 para fortalecer a Atenção Básica no Brasil. Em Goiânia (GO, foi implantado cinco equipes em uma região do município. O objetivo do estudo foi analisar o processo de implantação do Nasf em Goiânia a partir das percepções dos seus profissionais e gestores. Realizaram-se entrevistas, e os dados foram analisados a partir da técnica de análise temática. Conclui-se que o apoio matricial, a contratação de profissionais efetivos e a realização do processo formativo trouxeram avanços para o Nasf em Goiânia, embora com desafios frente às condições de trabalho e às resistências das Equipes de Saúde da Família.

  7. Tinjauan Kritis Simbol-simbol Budaya Nias dalam Iklan Produk Minuman Berenergi Studi Kasus Iklan Bermuatan Nilai Lokal Produk Kuku Bima Ener-G Versi “Ayo Wisata ke Sumatera” PT. Sido Muncul

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    Ariel Sarototonafo


    Full Text Available Symbols of local culture especially Nias on TVC (Television Commercial Kuku Bima Ener-G, version of “Let’s Travel to North Sumatra” from PT. Sidomuncul. Authors conducted a case study of the culture that campaigned Sidomuncul advertising through television media. The study was conducted using a critical research paradigm, which aim uncover Nias cultural symbols in the TVC of products Kuku Bima Ener-G PT Sidomuncul. The results suggested that the symbols used for Nias cultural values embodied in these symbols siding with the values espoused by Sidomuncul.

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

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    André Weiss

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

  9. Novel inexpensive fungi proteases: Production by solid state fermentation and characterization. (United States)

    Novelli, Paula Kern; Barros, Margarida Maria; Fleuri, Luciana Francisco


    A comparative study was carried out for proteases production using agroindustrial residues as substrate for solid state fermentation (SSF) of several fungal strains. High protease production was observed for most of the microorganisms studied, as well as very different biochemical characteristics, including activities at specific temperatures and a wide range of pH values. The enzymes produced were very different regarding optimum pH and they showed stability at 50 °C. Aspergillus oryzae showed stability at all pH values studied. Penicillium roquefortii and Aspergillus flavipes presented optimum activity at temperatures of 50 °C and 90 °C, respectively. Lyophilized protease from A. oryzae reached 1251.60 U/g and yield of 155010.66 U/kg of substrate. Therefore, the substrate as well as the microorganism strain can modify the biochemical character of the enzyme produced. The high protease activity and stability established plus the low cost of substrates, make these fungal proteases potential alternatives for the biotechnological industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Dynamic viscoelasticity of protease-treated rice batters for gluten-free rice bread making. (United States)

    Honda, Yuji; Inoue, Nanami; Sugimoto, Reina; Matsumoto, Kenji; Koda, Tomonori; Nishioka, Akihiro


    Papain (cysteine protease), subtilisin (Protin SD-AY10, serine protease), and bacillolysin (Protin SD-NY10, metallo protease) increased the specific volume of gluten-free rice breads by 19-63% compared to untreated bread. In contrast, Newlase F (aspartyl protease) did not expand the volume of the rice bread. In a rheological analysis, the viscoelastic properties of the gluten-free rice batters also depended on the protease categories. Principal component analysis (PCA) analysis suggested that the storage and loss moduli (G' and G″, respectively) at 35 °C, and the maximum values of G' and G″, were important factors in the volume expansion. Judging from the PCA of the viscoelastic parameters of the rice batters, papain and Protin SD-AY10 improved the viscoelasticity for gluten-free rice bread making, and Protin SD-NY effectively expanded the gluten-free rice bread. The rheological properties differed between Protin SD-NY and the other protease treatments.

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

    DEFF Research Database (Denmark)

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


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

  12. Production, purification and characterization of an aspartic protease from Aspergillus foetidus. (United States)

    Souza, Paula Monteiro; Werneck, Gabriela; Aliakbarian, Bahar; Siqueira, Felix; Ferreira Filho, Edivaldo Ximenes; Perego, Patrizia; Converti, Attilio; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa


    An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL -1 ) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g -1 ). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Introdução ao estudo da migração internacional na Amazônia Introduction to the study of international migration in the Amazon

    Directory of Open Access Journals (Sweden)

    Luis E Aragón


    Full Text Available Apesar da importância que a migração internacional alcançou na mídia e na academia no mundo inteiro, existem somente uns poucos estudos esparsos sobre a migração internacional na Amazônia. Como se apresenta o processo de migração internacional na Amazônia e qual é a sua importância nos destinos da região? Este artigo busca se aproximar da resposta a essa pergunta. O texto sintetiza resultados de investigações recentes realizadas pelo Grupo de Pesquisa Meio Ambiente, População e Desenvolvimento da Amazônia (MAPAZ, do Núcleo de Altos Estudos Amazônicos (NAEA da Universidade Federal do Pará. Primeiramente, destaca-se a dificuldade de se definir a Amazônia; posteriormente, são apresentadas algumas considerações sobre a migração internacional em nível da Grande Região, agregando os migrantes acumulados nas respectivas Amazônias nacionais, conforme os censos de população de cada país; e, finalmente, trata-se essa problemática no caso da Amazônia brasileira, destacando os países de nascimento e a distribuição espacial dos migrantes na Amazônia Legal. Na Amazônia, os padrões e as tendências discutidas aqui se tornarão mais dinâmicos e complexos em função do andamento de políticas de desenvolvimento na busca da integração regional e econômica.Despite the importance that international migration has attained in the media and academia worldwide, there are only a few scattered studies on international migration in the Amazon. How is the process of international migration presented in the Amazon and what is its importance in the destiny of the region? This article is an initial approach to that question. The text summarizes results of recent research conducted by the Research Group on Environment, Population and Development in the Amazon (MAPAZ of the Center for Advanced Amazonian Studies (NAEA of the Federal University of Pará. First, it discusses the difficulty of defining the Amazon, then some

  14. Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

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    Jeffrey W Koehler


    Full Text Available We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.


    NARCIS (Netherlands)



    The thermostability of neutral proteases has been shown to depend on autolysis which presumably occurs in flexible regions of the protein. In an attempt to rigidify such a region in the neutral protease of Bacillus stearothermophilus, residues in the solvent-exposed 63-69 loop were replaced by

  16. Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum. (United States)

    Castagliuolo, I; LaMont, J T; Nikulasson, S T; Pothoulakis, C


    Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans. PMID:8945570

  17. Chemical Tools for the Study of Intramembrane Proteases. (United States)

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


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

  18. A novel safety assessment strategy for non-intentionally added substances (NIAS) in carton food contact materials

    NARCIS (Netherlands)

    Koster, S.; Rennen, M.; Leeman, W.; Houben, G.; Muilwijk, B.; Acker, F. van; Krul, L.


    One of the main challenges in food contact materials research is to prove that the presence of non-intentionally added substances (NIAS) is not a safety issue. Migration extracts may contain many unknown substances present at low concentrations. It is difficult and time-consuming to identify all

  19. Survey on the use of psychotropic drugs by twelve military police units in the municipalities of Goiânia and Aparecida de Goiânia, state of Goiás, Brazil Pesquisa sobre uso de drogas psicotrópicas em 12 unidades da Polícia Militar nos municípios de Goiânia e Aparecida de Goiânia, Estado de Goiás, Brasil

    Directory of Open Access Journals (Sweden)

    Sérgio Henrique Nascente Costa


    Full Text Available OBJECTIVE: To determine the prevalence of psychotropic drug use among military police officers in the state of Goiás, Brazil. METHOD: Study carried out at twelve military police units located in the municipalities of Goiânia and Aparecida de Goiânia between March to October 2008. Volunteers (n = 221 were interviewed about drug use using a questionnaire especially designed by the Centro Brasileiro de Informações sobre Drogas Psicotrópicas (CEBRID. Descriptive statistics was used to determine the prevalence of licit and illicit drug use in the study sample. RESULTS: The frequency of use was divided into: 1 lifetime use: tobacco - 39.9%, alcohol - 87.8%, cannabis - 8.1%, cocaine - 1.8%, stimulants - 7.2%, solvents - 10.0%, sedatives, anxiolytics, antidepressants - 6.8%, LSD - 0.5%, Bentyl® - 0.5%, anabolic steroids - 5.4%; 2 use in the previous year: tobacco - 15.4%, alcohol - 72.9%, stimulants - 6.3%, solvents - 0.5%, sedatives, anxiolytics, antidepressants - 3.7%; 3 use in the previous 30 days: tobacco - 14.5%, alcohol - 57.5%, stimulants - 5.0%, solvents - 0.5, sedatives, anxiolytics, antidepressants - 3.7%. CONCLUSION: The high prevalence rate of psychotropic drug use found amoung military police officers in two cities of the state of Goiás in Brazil can be considered an important factor with potential influence on job activities.OBJETIVO: Verificar a prevalência do uso de drogas psicotrópicas por membros da Polícia Militar no Estado de Goiás, Brasil. MÉTODO: Estudo realizado de março a outubro de 2008 em 12 unidades da Polícia Militar dos municípios de Goiânia e Aparecida de Goiânia. Participantes voluntários (n = 221 foram entrevistados sobre uso de drogas utilizando-se questionário desenvolvido pelo Centro Brasileiro de Informações sobre Drogas Psicotrópicas (CEBRID. A estatística descritiva foi usada para determinar a prevalência de uso de drogas lícitas e ilícitas na amostra estudada. RESULTADOS: A frequência de

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

    African Journals Online (AJOL)

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

  1. Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

    DEFF Research Database (Denmark)

    Guarino, Carla; Hamon, Yveline; Croix, Cécile


    cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites....... These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases...

  2. Potent Inhibition of Feline Coronaviruses with Peptidyl Compounds Targeting Coronavirus 3C-like Protease (United States)

    Kim, Yunjeong; Mandadapu, Sivakoteswara Rao; Groutas, William C.; Chang, Kyeong-Ok


    Feline coronavirus infection is common among domestic and exotic felid species and usually associated with mild or asymptomatic enteritis; however, feline infectious peritonitis (FIP) is a fatal disease of cats that is caused by systemic infection with a feline infectious peritonitis virus (FIPV), a variant of feline enteric coronavirus (FECV). Currently, there is no specific treatment approved for FIP despite the importance of FIP as the leading infectious cause of death in young cats. During the replication process, coronavirus produces viral polyproteins that are processed into mature proteins by viral proteases, the main protease (3C-like [3CL] protease) and the papain-like protease. Since the cleavages of viral polyproteins are an essential step for virus replication, blockage of viral protease is an attractive target for therapeutic intervention. Previously, we reported the generation of broad-spectrum peptidyl inhibitors against viruses that possess a 3C or 3CL protease. In this study, we further evaluated the antiviral effects of the peptidyl inhibitors against feline coronaviruses, and investigated the interaction between our protease inhibitor and a cathepsin B inhibitor, an entry blocker, against feline coronaviruses in cell culture. Herein we report that our compounds behave as reversible, competitive inhibitors of 3CL protease, potently inhibited the replication of feline coronaviruses (EC50 in a nanomolar range) and, furthermore, the combination of cathepsin B and 3CL protease inhibitors led to a strong synergistic interaction against feline coronaviruses in cell culture systems. PMID:23219425

  3. Ubiquitin-specific protease 14 regulates cell proliferation and apoptosis in oral squamous cell carcinoma. (United States)

    Chen, Xiangyun; Wu, Jingjing; Chen, Yitian; Ye, Dongxia; Lei, Hu; Xu, Hanzhang; Yang, Li; Wu, Yingli; Gu, Wenli


    Ubiquitin-specific protease 14, a deubiquitinating enzyme, has been implicated in the tumorigenesis and progression of several cancers, but its role in oral squamous cell carcinoma remains to be elucidated. The aim of this study was to explore the expression pattern and roles of Ubiquitin-specific protease 14 in the occurrence and development of oral squamous cell carcinoma. Interestingly, Ubiquitin-specific protease 14 was overexpressed in oral cancer tissues and cell lines at both mRNA and protein levels. b-AP15, a specific inhibitor of Ubiquitin-specific protease 14, significantly inhibited the growth of cancer cells and increased cell apoptosis in a dose-dependent manner. Moreover, knockdown of Ubiquitin-specific protease 14 by shRNA significantly inhibited the proliferation and migration of cancer cells in vitro. Finally, using a xenograft mouse model of oral squamous cell carcinoma, knockdown of Ubiquitin-specific protease 14 markedly inhibited tumor growth and triggered the cancer cell apoptosis in vivo, supporting previous results. In conclusion, for the first time we have demonstrated the expression pattern of Ubiquitin-specific protease 14 in oral squamous cell carcinoma and verified a relationship with tumor growth and metastasis. These results may highlight new therapeutic strategies for tumor treatment, application of Ubiquitin-specific protease 14 selective inhibitor, such as b-AP15, or knockdown by shRNA. Collectively, Ubiquitin-specific protease 14 could be a potential therapeutic target for oral squamous cell carcinoma patients. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. Reflexões sobre hidrelétricas na Amazônia: água, energia e desenvolvimento

    Directory of Open Access Journals (Sweden)

    Bertha Koiffmann Becker

    Full Text Available O ensaio discute a implantação de hidrelétricas na Amazônia a partir das relações entre consumo de água e hidroeletricidade em diferentes escalas de análise, pois, se todos os lugares do planeta são afetados por processos globais, eles não o são de modo homogêneo. À escala global, domina o discurso apocalíptico de escassez crescente da água e do aquecimento global, exigindo a redução de emissões de gases de efeito estufa mediante o uso de energias renováveis e novas tecnologias. Na escala nacional, os problemas do Brasil são, pelo contrário, como gerir a abundância de água com justiça social e territorial, e como sustar a perda de 20% da energia elétrica produzida. Finalmente, é na escala regional - na Amazônia - que emergem os maiores problemas: i o maior paradoxo entre abundância de água e inacessibilidade social ao recurso; ii grande parte das hidrelétricas planejadas para o país será ali construída, com risco dos graves impactos conhecidos; iii a cogitada obrigatoriedade da construção de eclusas em todas as hidrelétricas, proposta pelo setor industrial em benefício da navegabilidade dos rios, deverá propiciar, na verdade, o escoamento de commodities produzidas na região Centro-Oeste. Coloca-se, assim, para a sociedade e para o governo, a questão ética se realmente são necessárias tantas hidrelétricas na Amazônia.

  5. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines. (United States)

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas


    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Protease of Stenotrophomonas sp. from Indonesian fermented food: gene cloning and analysis

    Directory of Open Access Journals (Sweden)

    Frans Kurnia


    Full Text Available Screening of proteolytic and fibrinolytic bacteria from Indonesian soy bean based fermented food Oncom revealed several potential isolates. Based on 16s rDNA gene analysis, one particular isolate with the highest proteolytic and fibrinolytic activity was identified as Stenotrophomonas sp. The protease gene was amplified to generate a 1749 bp Polymerase Chain Reaction product and BLAST analysis, revealed 90% homology with gene encoding protease enzyme from Stenotrophomonas maltophilia. The putative amino acid sequence indicated a serine protease enzyme with typical amino acid aspartate, histidine and serine in the catalytic triad. The gene was translated into a pre-pro-protein consisted of cleavage site on its N terminal and Pre-Peptidase Cterminal domain. Cloning of the protease gene in pET22b with Escherichia coli BL21 DE3 as the host showed that the gene was expressed as insoluble protein fraction. This is the first report for analysis of protease gene from food origin Stenotrophomonas sp.

  7. The kunitz protease inhibitor form of the amyloid precursor protein (KPI/APP) inhibits the proneuropeptide processing enzyme prohormone thiol protease (PTP). Colocalization of KPI/APP and PTP in secretory vesicles. (United States)

    Hook, V Y; Sei, C; Yasothornsrikul, S; Toneff, T; Kang, Y H; Efthimiopoulos, S; Robakis, N K; Van Nostrand, W


    Proteolytic processing of proenkephalin and proneuropeptides is required for the production of active neurotransmitters and peptide hormones. Variations in the extent of proenkephalin processing in vivo suggest involvement of endogenous protease inhibitors. This study demonstrates that "protease nexin 2 (PN2)," the secreted form of the kunitz protease inhibitor (KPI) of the amyloid precursor protein (APP), potently inhibited the proenkephalin processing enzyme known as prohormone thiol protease (PTP), with a Ki,app of 400 nM. Moreover, PTP and PN2 formed SDS-stable complexes that are typical of kunitz protease inhibitor interactions with target proteases. In vivo, KPI/APP (120 kDa), as well as a truncated form of KPI/APP that resembles PN2 in apparent molecular mass (110 kDa), were colocalized with PTP and (Met)enkephalin in secretory vesicles of adrenal medulla (chromaffin granules). KPI/APP (110-120 kDa) was also detected in pituitary secretory vesicles that contain PTP. In chromaffin cells, calcium-dependent secretion of KPI/APP with PTP and (Met)enkephalin demonstrated the colocalization of these components in functional secretory vesicles. These results suggest a role for KPI/APP inhibition of PTP in regulated secretory vesicles. In addition, these results are the first to identify an endogenous protease target of KPI/APP, which is developmentally regulated in aging and Alzheimer's disease.

  8. Photoionization and Recombination (United States)

    Nahar, Sultana N.


    Theoretically self-consistent calculations for photoionization and (e + ion) recombination are described. The same eigenfunction expansion for the ion is employed in coupled channel calculations for both processes, thus ensuring consistency between cross sections and rates. The theoretical treatment of (e + ion) recombination subsumes both the non-resonant recombination ("radiative recombination"), and the resonant recombination ("di-electronic recombination") processes in a unified scheme. In addition to the total, unified recombination rates, level-specific recombination rates and photoionization cross sections are obtained for a large number of atomic levels. Both relativistic Breit-Pauli, and non-relativistic LS coupling, calculations are carried out in the close coupling approximation using the R-matrix method. Although the calculations are computationally intensive, they yield nearly all photoionization and recombination parameters needed for astrophysical photoionization models with higher precision than hitherto possible, estimated at about 10-20% from comparison with experimentally available data (including experimentally derived DR rates). Results are electronically available for over 40 atoms and ions. Photoionization and recombination of He-, and Li-like C and Fe are described for X-ray modeling. The unified method yields total and complete (e+ion) recombination rate coefficients, that can not otherwise be obtained theoretically or experimentally.

  9. As redes para além dos rios: urbanização e desequilíbrios na Amazônia Brasileira


    Douglas Sathler dos Reis


    Nas últimas décadas, as redes urbanas que se estendem sobre a Amazônia Legal têm evoluído com o surgimento de cidades de médio porte e com a multiplicação de pequenas aglomerações urbanas, que seguem os traçados das principais rodovias e rios da região. Em um contexto de transformações globais, com aparecimento das Redes Móveis em meio às novas formas de flexibilidade e interatividade, as redes da Amazônia Legal apresentam diversos aspectos que devem ser estudados sem euforia, diante de uma s...

  10. Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs.

    Directory of Open Access Journals (Sweden)

    Alex Loukas


    Full Text Available Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1.We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056 and fecal egg counts (p = 0.018 in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049 and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasite's ability to digest blood.To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine.

  11. Intoxicações por plantas em ruminantes e equídeos na região central de Rondônia Plant poisonings in ruminants and equidae in central region of Rondônia state, Northern Brazil

    Directory of Open Access Journals (Sweden)

    Sandro Vargas Schons


    Full Text Available Foi realizado um levantamento em 12 municípios da região central de Rondônia sobre a presença de plantas tóxicas e ocorrência de surtos de intoxicação em ruminantes e equídeos. O trabalho foi desenvolvido mediante a utilização de um questionário aplicado a médicos veterinários, agrônomos, zootecnistas e produtores rurais, com o objetivo de identificar as principais plantas tóxicas que ocorrem na região. Trinta e quatro entrevistados relataram casos de intoxicação por uma ou mais plantas comprovadamente tóxicas como: Palicourea marcgravii (12 surtos, Palicourea grandiflora e Enterolobium contortisiliquum (sete surtos cada e Palicourea juruana, Brachiaria radicans, Brachiaria brizantha e Manihot esculenta (dois surtos cada. Em ovinos, foram relatados dois surtos de fotossensiblização por Brachiaria decumbens e um surto de mortalidade por Palicourea grandiflora. Dos 34 surtos relatados em bovinos pelos entrevistados, 374 (8,9% animais foram afetados e 311 (7,4% morreram, de um total de 4.192 de ambos os sexos sob risco. De um total de 250 ovinos sob risco, três surtos de intoxicação por plantas foram relatados e afetaram 28 animais, dos quais 20 morreram. Amorimia sp., previamente desconhecida como tóxica, foi identificada como causa de morte súbita em 32% das propriedades. Quinze surtos de cólica em equídeos que pastavam cultivares de Panicum maximum ('Massai', 'Tanzânia' e 'Mombaça' durante o período das chuvas foram, também, observados. Os resultados do presente trabalho demonstram a importância significativa das intoxicações por plantas como causa de perdas econômicas para a pecuária da região central do Estado de Rondônia. Com a realização deste trabalho, o número de plantas tóxicas para ruminantes com a confirmação de ocorrência de surtos com mortalidade na região passou de um para nove, o que confirma que um trabalho sistemático de investigação é necessário para o conhecimento da import

  12. Purification of two high molecular weight proteases from rabbit reticulocyte lysate

    International Nuclear Information System (INIS)

    Hough, R.; Pratt, G.; Rechsteiner, M.


    The authors have purified two large proteases from rabbit reticulocyte lysate. The enzymes are so similar in their chromatographic behavior that each is the only significant contaminant of the other during the final stages of purification. At pH 7.8, both hydrolyze 125 I-α-casein and 4-methylcoumaryl-7-amide (MCA) derivatives with tyrosine, phenylalanine or arginine at the P 1 position. The larger, ATP-dependent enzyme degrades ubiquitin-lysozyme conjugates, but it does not degrade unmodified lysozyme. Hydrolysis of Suc-Leu-Leu-Val-Tyr-MCA by this enzyme is also stimulated two-fold in the presence of ATP. The protease has a molecular weight of 950,000 based on sedimentation, gel filtration and non-denaturing PAGE. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that the protease is composed of a number of subunits with molecular masses between 32 and 110 kDa. Densitometric analysis showed equivalent amounts of the two larger chains, and the presence of one copy of each in the native enzyme would be consistent with an M/sub r/ of 950,000. The smaller protease has a molecular weight of 700,000 and is composed of 8 to 10 subunits ranging from 21,000 to 32,000. It cleaves ubiquitin-lysozyme conjugates only slightly, and hydrolysis of conjugates or fluorogenic peptide substrates is not stimulated by ATP. This protease appears similar, if not identical, to the multicatalytic protease complex first purified by Wilk and Orlowski

  13. Enhanced Productivity of Serine Alkaline Protease by Bacillus sp. Using Soybean as Substrate

    Directory of Open Access Journals (Sweden)

    Saurabh, S.


    Full Text Available The growth and protease production by Bacillus sp. (SBP-29 was examined for poultry processing industries. The maximum protease activity was 3028 U/mL using 1.5% (w/v of soybean meal as substrate. Soybean meal is an inexpensive and readily available, thus it can be used as the cost effective crude material for the production of an extracellular protease. Inorganic nitrogen sources proved to be less favorable, for protease production as strong catabolic repression was observed with ammonium ions. A maximum of 3208 U/mL of protease was produced in 18 h in a 10L bioreactor. The enzyme has temperature and pH optima of 60°C and 9.5 respectively. However, the temperature stability range is from 20-90 °C and pH stability range is from 6.0–12.0. The protease was completely inhibited by phenylmethylsulfonyl fluoride (PMSF and diodopropyl fluorophosphate (DFP, with little increase (10-15% in the production of upon addition of Ca++ and Mg++.

  14. Interfacial behavior of alkaline protease at the air-water and oil-water interfaces (United States)

    Zhang, Jian; Li, Yanyan; Wang, Jing; Zhang, Yue


    The interfacial behavior of alkaline protease at the air-water and n-hexane-water interfaces was investigated using interfacial tension, dilatational rheology and dynamic light scattering. Additionally, different adsorption models which are Langmuir, Frumkin, Reorientation-A and Reorientation-R were used to fitting the data of equilibrium interfacial tension for further understanding the interfacial behavior of alkaline protease. Data fitting of the equilibrium interfacial tension was achieved by IsoFit software. The results show that the molecules arrangement of the alkaline protease at the n-hexane-water interface is more tightly than at the air-water interface. The data were further analyzed to indicate that the hydrophobic chains of alkaline protease penetrate into oil phase deeper than the air phase. Also data indicate that the electrostatic interactions and hydrophobic interactions at the n-hexane-water interface are stronger than at the air-water interface within molecules of the alkaline protease. Based on comprehensive analysis of the adsorption kinetics and interfacial rheological properties, interfacial structures mechanism of alkaline protease at n-hexane-water and air-water interfaces was proposed.

  15. Characterization of an alkaline protease associated with a granulosis virus of Plodia interpunctella. (United States)

    Tweeten, K A; Bulla, L A; Consigli, R A


    An alkaline protease was found to be associated with the granulosis virus of the Indian meal moth. Plodia interpunctella. The protease was located within the protein matrix of the occluded virus and hydrolyzed the major constituent of this matrix, a 28,000-dalton protein (granulin), to a mixture of polypeptides ranging in molecular weight from 10,000 to 27,000. A rapid, sensitive assay for the protease was developed using radioactively labeled granulosis virus as substrate. With this assay, the proteolytic activity could be detected by measuring the release of acid-soluble peptides from the labeled virus. The protease had a pH optimum of 10.5 and a temperature optimum of 40 degrees C and was inhibited by diisopropyl phosphorofluoridate, phenylmethylsulfonyl fluoride, and L-(1-tosylamido-2-phenyl) ethyl chloromethyl ketone. Purification of the protease from matrix protein was achieved by anion-exchange and gel permeation chromatography. The molecular weight of the isolated protease, determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, was approximately 14,000.


    Directory of Open Access Journals (Sweden)



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

  17. Soluble expression and purification of the recombinant bioactive peptide precursor BPP-1 in Escherichia coli using a cELP-SUMO dual fusion system. (United States)

    Rao, Shengqi; Zang, Xiangyu; Yang, Zhenquan; Gao, Lu; Yin, Yongqi; Fang, Weiming


    A bioactive peptide precursor (BPP-1, 14.3 kDa/115AA), a newly designed polypeptide that may exert a potential antihypertensive effect in vivo, is composed of many different ACE inhibitory peptides and antioxidant peptides tandemly linked according to the restriction sites of gastrointestinal proteases. In this report, we present a novel method to obtain soluble BPP-1 in Escherichia coli using cationic elastin-like polypeptide and SUMO (cELP-SUMO) tags. The cELP-SUMO-tagged fusion protein was expressed in soluble form at 20 °C for 20 h. After purification based on the inverse transition cycling (ITC) method, the purified cELP-SUMO-CFPP fusion protein was subsequently cleaved by a SUMO protease to release the mature BPP-1. After a subsequent simple salt precipitation process, approximately 167.2 mg of recombinant BPP-1 was obtained from 1 l of bacterial culture with at least 92% purity. The molecular mass (Mr) of the recombinant BPP-1 was confirmed by MALDI-TOF MS to equal 14,347. The purified BPP-1 was subjected to simulated gastrointestinal digestion, and the resulting hydrolysates exhibited notable ACE inhibitory and antioxidant activities in vitro. This report provides the first description of the soluble production of a bioactive peptide multimer with potential ACE inhibitory and antioxidant activities in E. coli using a cELP-SUMO tag. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Characterization and milk coagulating properties of Cynanchum otophyllum Schneid. proteases. (United States)

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


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

  19. The action of neutrophil serine proteases on elastin and its precursor

    DEFF Research Database (Denmark)

    Heinz, Andrea; Jung, Michael C; Jahreis, Günther


    This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass...... spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three...... proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr...

  20. Barley (Hordeum vulgare L.) cysteine proteases: heterologous expression, purification and characterization

    DEFF Research Database (Denmark)

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


    During germination of barley seeds, mobilization of protein is essential and cysteine proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins. Cysteine proteases exist as pro-enzyme and is activated through reduction of the active...... site cysteines and by removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. A cDNA clone of the barley key cysteine endoprotease...

  1. Effectiveness of Ritonavir-Boosted Protease Inhibitor Monotherapy in Clinical Practice Even with Previous Virological Failures to Protease Inhibitor-Based Regimens.

    Directory of Open Access Journals (Sweden)

    Luis F López-Cortés

    Full Text Available Significant controversy still exists about ritonavir-boosted protease inhibitor monotherapy (mtPI/rtv as a simplification strategy that is used up to now to treat patients that have not experienced previous virological failure (VF while on protease inhibitor (PI -based regimens. We have evaluated the effectiveness of two mtPI/rtv regimens in an actual clinical practice setting, including patients that had experienced previous VF with PI-based regimens.This retrospective study analyzed 1060 HIV-infected patients with undetectable viremia that were switched to lopinavir/ritonavir or darunavir/ritonavir monotherapy. In cases in which the patient had previously experienced VF while on a PI-based regimen, the lack of major HIV protease resistance mutations to lopinavir or darunavir, respectively, was mandatory. The primary endpoint of this study was the percentage of participants with virological suppression after 96 weeks according to intention-to-treat analysis (non-complete/missing = failure.A total of 1060 patients were analyzed, including 205 with previous VF while on PI-based regimens, 90 of whom were on complex therapies due to extensive resistance. The rates of treatment effectiveness (intention-to-treat analysis and virological efficacy (on-treatment analysis at week 96 were 79.3% (CI95, 76.8-81.8 and 91.5% (CI95, 89.6-93.4, respectively. No relationships were found between VF and earlier VF while on PI-based regimens, the presence of major or minor protease resistance mutations, the previous time on viral suppression, CD4+ T-cell nadir, and HCV-coinfection. Genotypic resistance tests were available in 49 out of the 74 patients with VFs and only four patients presented new major protease resistance mutations.Switching to mtPI/rtv achieves sustained virological control in most patients, even in those with previous VF on PI-based regimens as long as no major resistance mutations are present for the administered drug.

  2. Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation (United States)

    Liu, Xiaoqing; Xiu, Zhilong; Hao, Ce


    Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79's loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50'. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1' subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2' subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design.

  3. Studies to Prevent Degradation of Recombinant Fc-Fusion Protein Expressed in Mammalian Cell Line and Protein Characterization

    Directory of Open Access Journals (Sweden)

    Sanjukta Chakrabarti


    Full Text Available Clipping of recombinant proteins is a major issue in animal cell cultures. A recombinant Fc-fusion protein, VEGFR1(D1–D3-Fc expressed in CHOK1SV GS-KO cells was observed to be undergoing clippings in lab scale cultures. Partial cleaving of expressed protein initiated early on in cell culture and was observed to increase over time in culture and also on storage. In this study, a few parameters were explored in a bid to inhibit clipping in the fusion protein The effects of culture temperature, duration of culture, the addition of an anti-clumping agent, ferric citrate and use of protease inhibitor cocktail on inhibition of proteolysis of the Fc fusion were studied. Lowering of culture temperature from 37 to 30 °C alone appears to be the best solution for reducing protein degradation from the quality, cost and regulatory points of view. The obtained Fc protein was characterized and found to be in its stable folded state, exhibiting a high affinity for its ligand and also biological and functional activities.

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

    International Nuclear Information System (INIS)

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


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

  5. Corruption of innate immunity by bacterial proteases. (United States)

    Potempa, Jan; Pike, Robert N


    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.

  6. Crescimento e estado nutricional de helicônia irrigada com água residuária tratada em casa de vegetação Growth and nutritional status of helicônia irrigated with treated wastewater in greenhouse

    Directory of Open Access Journals (Sweden)

    Olívia S. N. Santos


    Full Text Available Neste trabalho se estudaram o desenvolvimento e o estado nutricional de helicônias irrigadas com água residuária tratada de origem doméstica associada a fertilizantes químicos (NPK. O experimento foi conduzido em casa de vegetação na qual se utilizaram plantas de helicônia Golden Adrian. O experimento foi instalado no esquema fatorial 5 x 3 + 1 constituídos de cinco níveis de lâmina de água aplicada (equivalente a 60, 80, 100, 120 e 140% da evaporação do tanque classe A ECA, três tipos de água (água residuária (AR, água pluvial (AP e mistura de 50% de AR + 50% de AP e testemunha (solo sem adubação e recebendo 100% da ECA de água pluvial. O delineamento experimental utilizado foi blocos ao acaso, com cinco repetições. A utilização de AR e as lâminas maiores proporcionaram acréscimo nos parâmetros de crescimento avaliados. Os teores de macronutrientes presentes nas folhas das plantas irrigadas com AR se comportaram de forma semelhante às plantas que receberam AP. O uso de AR e AP resultou em acréscimo de teores de micronutrientes nas folhas, principalmente Fe e Mn, acima dos teores recomendados para a cultura.In this study the growth and nutritional status of helicônias irrigated with treated wastewater of domestic origin associated with chemical fertilizers (NPK was studied. The experiment was conducted in a greenhouse utilizing plants of helicônia Golden Adrian. The experiment was installed in 5 x 3 + 1 factorial scheme consisting of five depths of irrigation water (equivalent to 60, 80, 100, 120 and 140% of class A pan evaporation - ECA, three types of water (wastewater (AR, rainwater (AP and a mixture of 50% AR + 50% AP and control (soil without fertilizer and receiving rainwater corresponding to 100% ECA. The experimental design was in randomized blocks with five repetitions. The use of AR and larger depths of irrigation provided increases in evaluated parameters of growth. The levels of macronutrients in the

  7. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

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

  8. Carbohydrase and protease supplementation increased ...

    African Journals Online (AJOL)

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

  9. Molecular models of NS3 protease variants of the Hepatitis C virus

    Directory of Open Access Journals (Sweden)

    Mello Isabel MVGC


    Full Text Available Abstract Background Hepatitis C virus (HCV currently infects approximately three percent of the world population. In view of the lack of vaccines against HCV, there is an urgent need for an efficient treatment of the disease by an effective antiviral drug. Rational drug design has not been the primary way for discovering major therapeutics. Nevertheless, there are reports of success in the development of inhibitor using a structure-based approach. One of the possible targets for drug development against HCV is the NS3 protease variants. Based on the three-dimensional structure of these variants we expect to identify new NS3 protease inhibitors. In order to speed up the modeling process all NS3 protease variant models were generated in a Beowulf cluster. The potential of the structural bioinformatics for development of new antiviral drugs is discussed. Results The atomic coordinates of crystallographic structure 1CU1 and 1DY9 were used as starting model for modeling of the NS3 protease variant structures. The NS3 protease variant structures are composed of six subdomains, which occur in sequence along the polypeptide chain. The protease domain exhibits the dual beta-barrel fold that is common among members of the chymotrypsin serine protease family. The helicase domain contains two structurally related beta-alpha-beta subdomains and a third subdomain of seven helices and three short beta strands. The latter domain is usually referred to as the helicase alpha-helical subdomain. The rmsd value of bond lengths and bond angles, the average G-factor and Verify 3D values are presented for NS3 protease variant structures. Conclusions This project increases the certainty that homology modeling is an useful tool in structural biology and that it can be very valuable in annotating genome sequence information and contributing to structural and functional genomics from virus. The structural models will be used to guide future efforts in the structure

  10. An antibiotic, heavy metal resistant and halotolerant Bacillus cereus SIU1 and its thermoalkaline protease

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    Vikram Surendra


    Full Text Available Abstract Background Many workers have reported halotolerant bacteria from saline conditions capable of protease production. However, antibiotic resistance and heavy metal tolerance pattern of such organisms is not documented very well. Similarly, only a few researchers have reported the pattern of pH change of fermentation medium during the course of protease production. In this study, we have isolated a halotolerant Bacillus cereus SIU1 strain from a non-saline environment and studied its antibiotic and heavy metal resistance pattern. The isolate produces a thermoalkaline protease and changes the medium pH during the course of fermentation. Thermostability of protease was also studied for 30 min. Results Seventy bacterial strains isolated from the soils of Eastern Uttar Pradesh, India were screened for protease production. All of them exhibited protease activity. However, 40% bacterial isolates were found good protease producers as observed by caseinolytic zones on milk agar plates. Among them, culture S-4 was adjudged as the best protease producer, and was identified as Bacillus cereus by morphological, biochemical and 16 S rDNA sequence analyses. The isolate was resistant to heavy metals (As2+, Pb2+, Cs1+ and antibiotics (penicillin, lincomycin, cloxacillin, pefloxacin. Its growth behavior and protease production was studied at 45°C and pH 9.0. The protease units of 88 ml-1 were noted in unoptimized modified glucose yeast extract (GYE medium during early stationary phase at 20 h incubation period. The enzyme was stable in the temperature range of 35°-55°C. Conclusions An antibiotic and heavy metal resistant, halotolerant Bacillus cereus isolate is capable of producing thermoalkaline protease, which is active and stable at pH 9.0 and 35°-55°C. This isolate may be useful in several industrial applications owing to its halotolerance and antibiotic and heavy metal resistance characteristics.

  11. Improvement of shelf life of soymilk using immobilized protease of Oerskovia xanthineolytica NCIM 2839. (United States)

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


    Protease enzyme has lot of commercial applications, so the cost-effective production of protease using sunflower oil seed waste was carried out from Oerskovia xanthineolyitca NCIM 2839. The maximum protease production was after 24 h of incubation with 2.5 % oil seed waste concentration. O. xanthineolytica was found to produce two proteases-P1 and P2. The proteases were purified using 60 % cold acetone precipitation and DEAE-cellulose ion exchange chromatography. SDS-PAGE revealed molecular weight of P1 and P2 was 36 and 24 kDa, respectively. P1 and P2 were optimally active at pH 7.0 and pH 7.5 at temperature 35 and 40 °C, respectively. Analysis of hydrolyzed product of P1 and P2 by HPLC reveals that the P1 has endoprotease and P2 has exoprotease activity. The treated soy milk with immobilized proteases showed increased shelf life and removal of off flavor.

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

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    Landys A. Lopez Quezada


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

  13. Chernobyl and Goiânia lessons for responding to radiological terrorism. (United States)

    Steinhausler, Friedrich


    The deployment of a radiological dispersal device (RDD) is likely to result in relatively low radiation exposure of the targeted population, insufficient to cause a severe radiation detriment. Nevertheless, due to atmospheric dispersion of the radioactive material, an urban area equaling several city blocks could be affected. The current knowledge base concerning the response to radiological terrorism, focusing mainly on environmental cleanup and site recovery (CSR) of areas with radioactive contamination due to the deployment of an RDD, is largely derived from military scientific tests or exercises assembled over the past 50 y with only limited applicability to the consequences of an RDD detonating in a city. This paper focuses on the extensive experience in CSR gained in the management of the radiological accident contaminating the Brazilian city of Goiânia in 1987, and managing the aftermath of the Chernobyl reactor accident in 1986. The incident in Goiânia demonstrated the numerous practical difficulties of implementing a sound CSR, based on a balanced judgment of all relevant factors, such as radiation safety, environmental issues, economic consequences, and public fear. A review of the different stages of the intervention policy in the former Soviet Union reveals that risk-benefit cost analysis was not used for the decision-making process during the later stages of the post-accident situation. Instead, a CSR policy was adopted that resulted in continuously escalating costs. The results of this analysis are used to develop an Integrated Cleanup and Site Restoration Concept and recommend practically applicable solutions from Lessons Learned.

  14. Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side. (United States)

    Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A


    The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors

  15. Design of novel HIV-1 protease inhibitors incorporating isophthalamide-derived P2-P3 ligands: Synthesis, biological evaluation and X-ray structural studies of inhibitor-HIV-1 protease complex

    Energy Technology Data Exchange (ETDEWEB)

    Ghosh, Arun K.; Brindisi, Margherita; Nyalapatla, Prasanth R.; Takayama, Jun; Ella-Menye, Jean-Rene; Yashchuk, Sofiya; Agniswamy, Johnson; Wang, Yuan-Fang; Aoki, Manabu; Amano, Masayuki; Weber, Irene T.; Mitsuya, Hiroaki


    Based upon molecular insights from the X-ray structures of inhibitor-bound HIV-1 protease complexes, we have designed a series of isophthalamide-derived inhibitors incorporating substituted pyrrolidines, piperidines and thiazolidines as P2-P3 ligands for specific interactions in the S2-S3 extended site. Compound 4b has shown an enzyme Ki of 0.025 nM and antiviral IC50 of 69 nM. An X-ray crystal structure of inhibitor 4b-HIV-1 protease complex was determined at 1.33 Å resolution. We have also determined X-ray structure of 3b-bound HIV-1 protease at 1.27 Å resolution. These structures revealed important molecular insight into the inhibitor–HIV-1 protease interactions in the active site.

  16. Telejornalismo na Amaz?nia: o FTP como instrumento de integra??o regional


    Batista, Lu?s Augusto Pires


    Esta disserta??o investiga o efeito do sistema FTP (File Transfer Protocol ou Protocolo de Transfer?ncia de Arquivo) no processo de produ??o do telejornalismo das emissoras da Rede Amaz?nica (TV Amazonas, TV Rond?nia, TV Acre, TV Roraima e TV Amap?), a partir da Central de Jornalismo sediada na capital do estado no Amazonas e seus efeitos no cotidiano da comunidade do munic?pio de Manacapuru, que passou a ser n?o apenas consumidora de informa??o, mas tamb?m produtora dela. C...

  17. An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I. (United States)

    Le, Tien; Lee, Hak Jin; Jin, Hyung Jong


    A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Cenários de desmatamento para a Amazônia

    Directory of Open Access Journals (Sweden)

    Britaldo Silveira Soares-Filho


    Full Text Available A AMAZÔNIA está entrando em uma era de rápidas mudanças impulsionadas pela previsão de asfaltamento de rodovias que estimularão a expansão da fronteira agrícola e de exploração madeireira. O declínio do custo de transporte tem importantes implicações para a biodiversidade, emissão de gases que contribuem para o efeito estufa e prosperidade da sociedade da Amazônia a longo prazo. Para analisar esse contexto, foi desenvolvido um modelo de simulação de desmatamento na bacia Amazônica, sensível a diferentes cenários de políticas públicas frente à expansão da infra-estrutura de transporte pela região. Resultados do modelo indicam que, dentro de um cenário pessimista, o desmatamento projetado pode eliminar, até meados deste século, 40% dos atuais 5,4 milhões de km² de florestas da Amazônia, liberando o equivalente a 32 Pg (10(9 toneladas de carbono para atmosfera. A modelagem de cenários alternativos aponta que a expansão de uma rede de áreas protegidas, efetivamente implementadas, poderia reduzir em até 1/3 as perdas florestais projetadas. Contudo, outras medidas de conservação são ainda necessárias para se manter a integridade funcional das paisagens e bacias hidrográficas amazônicas. Atuais experimentos em conservação florestal em propriedades privadas, mercados de serviços ambientais e zoneamento agro-ecológico devem ser refinados e multiplicados a fim de se buscar uma conservação extensiva.THE AMAZON is entering an era of rapid changes as new transportation corridors traverse the region, stimulating the expansion of logging and agricultural frontiers. The declining cost of transportation has important implications for biodiversity, greenhouse gas emissions, and the long-term prosperity of the Amazon society. To analyze this context, we have developed an empirically based, policy-sensitive model of deforestation for the Amazon basin. Model output for the worst-case scenario shows that, by 2050

  19. Purification and characterization of a serine protease (CESP) from mature coconut endosperm (United States)

    Panicker, Leelamma M; Usha, Rajamma; Roy, Samir; Mandal, Chhabinath


    Background In plants, proteases execute an important role in the overall process of protein turnover during seed development, germination and senescence. The limited knowledge on the proteolytic machinery that operates during seed development in coconut (Cocos nucifera L.) prompted us to search for proteases in the coconut endosperm. Findings We have identified and purified a coconut endosperm protease (CESP) to apparent homogeneity. CESP is a single polypeptide enzyme of approximate molecular mass of 68 kDa and possesses pH optimum of 8.5 for the hydrolysis of BAPNA. Studies relating to substrate specificity and pattern of inhibition by various protease inhibitors indicated that CESP is a serine protease with cleavage specificity to peptide bonds after arginine. Purified CESP was often autolysed to two polypeptides of 41.6 kDa (CESP1) and 26.7 kDa (CESP2) and is confirmed by immunochemistry. We have shown the expression of CESP in all varieties of coconut and in all stages of coconut endosperm development with maximum amount in fully matured coconut. Conclusion Since the involvement of proteases in the processing of pre-proteins and maintenance of intracellular protein levels in seeds are well known, we suspect this CESP might play an important role in the coconut endosperm development. However this need to be confirmed using further studies. PMID:19426537

  20. Estratégia analítica para determinação de nitrito e amônia empregando spot test e análise de imagem digital

    Directory of Open Access Journals (Sweden)

    J. L. O. Santos


    Full Text Available Neste trabalho é proposta uma estratégia simples para determinação simultânea de nitrito e amônia empregando spot test e imagem digital. O método é baseado na reação do nitrito com a sulfanilamida, formando um cátion de diazônio que posteriormente é acoplado ao dicloreto de N-(1-nafitil etilenodiamina formando uma diazo composto de coloração rósea. A determinação da amônia foi realizada empregando a reação de berthelot modificada, onde o íon amônio em presença de salicilato, nitroprussiato e hipoclorito reage formando um composto de coloração verde. Os sinais analíticos foram obtidos a partir das intensidades de coloração por imagem digital no spot test do produto da reação. Após otimizações do sistema de análise e detecção, as seguintes figuras de mérito foram obtidas: concentração linear entre 0,2 a 10,0 mg L-1 para nitrito e 1,0 a 10,0 mg L-1 para a amônia. Os limites de detecção para o nitrito e amônia foram 0,05 mg L-1 e 0,27 mg L-1, respectivamente. A estratégia proposta foi aplicada com sucesso na determinação de nitrito e amônia em amostras de água subterrânea da zona urbana da cidade de Barreiras-Ba.

  1. Chemistry and biology of natural product derived protease inhibitors


    Stolze, Sara Christina


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

  2. Epidemiological features of rotavirus infection in Goiânia, Goiás, Brazil, from 1986 to 2000

    Directory of Open Access Journals (Sweden)

    Divina das Dôres de Paula Cardoso


    Full Text Available A total of 2,605 faecal specimens from children up to 10 years old with or without diarrhoea were collected. Samples were obtained from 1986 to 2000 in hospitals, outpatient clinics and day-care centers in Goiânia, Goiás. Two methodologies for viral detection were utilized: a combined enzyme immunoassay for rotavirus and adenovirus and polyacrylamide gel electrophoresis. Results showed 374 (14.4% faecal specimens positive for Rotavirus A, most of them collected from hospitalized children. A significant detection rate of rotavirus during the period from April to August, dry season in Goiânia, and different frequencies of viral detection throughout the years of study were also observed. Rotavirus was significantly related to hospitalization and to diarrhoeal illness in children up to 24 months old. This study reinforces the importance of rotavirus as a cause of diarrhoea in children and may be important in regards to the implementation of rotavirus vaccination strategies in our country.

  3. Allergenic proteases cleave the chemokine CX3CL1 directly from the surface of airway epithelium and augment the effect of rhinovirus. (United States)

    Loxham, M; Smart, D E; Bedke, N J; Smithers, N P; Filippi, I; Blume, C; Swindle, E J; Tariq, K; Howarth, P H; Holgate, S T; Davies, D E


    CX3CL1 has been implicated in allergen-induced airway CD4 + T-lymphocyte recruitment in asthma. As epidemiological evidence supports a viral infection-allergen synergy in asthma exacerbations, we postulated that rhinovirus (RV) infection in the presence of allergen augments epithelial CX3CL1 release. Fully differentiated primary bronchial epithelial cultures were pretreated apically with house dust mite (HDM) extract and infected with rhinovirus-16 (RV16). CX3CL1 was measured by enzyme-linked immunosorbent assay and western blotting, and shedding mechanisms assessed using inhibitors, protease-activated receptor-2 (PAR-2) agonist, and recombinant CX3CL1-expressing HEK293T cells. Basolateral CX3CL1 release was unaffected by HDM but stimulated by RV16; inhibition by fluticasone or GM6001 implicated nuclear factor-κB and ADAM (A Disintegrin and Metalloproteinase) sheddases. Conversely, apical CX3CL1 shedding was stimulated by HDM and augmented by RV16. Although fluticasone or GM6001 reduced RV16+HDM-induced apical CX3CL1 release, heat inactivation or cysteine protease inhibition completely blocked CX3CL1 shedding. The HDM effect was via enzymatic cleavage of CX3CL1, not PAR-2 activation, yielding a product mitogenic for smooth muscle cells. Extracts of Alternaria fungus caused similar CX3CL1 shedding. We have identified a novel mechanism whereby allergenic proteases cleave CX3CL1 from the apical epithelial surface to yield a biologically active product. RV16 infection augmented HDM-induced CX3CL1 shedding-this may contribute to synergy between allergen exposure and RV infection in triggering asthma exacerbations and airway remodeling.

  4. Perdas de colônias de Apis mellifera L. no inverno suplementadas com alimentação artificial com pólen e favos de mel

    Directory of Open Access Journals (Sweden)

    Adhemar Pegoraro


    Full Text Available O objetivo deste trabalho foi avaliar o efeito do tipo de suplementação, nas áreas de cria, alimento e infestação por Varroa destructor nas colônias perdidas. Foram analisadas18 colônias de Apis mellifera L. alojadas em colméias modelo Langstroth e submetidas a dois tipos de suplementação alimentar: 1- cremosa (açúcar cristal moído, mel e pólen; 2- favos de mel. As áreas de ovo-larva e pupa, néctar, mel e pólen das 18 colônias foram fotografadas e analisadas com o auxílio do software AutoCAD. As colônias perdidas suplementadas com alimento artificial e pólen possuíam áreas de: ovo-larva (87,79 ± 70,38cm2, pupa (228,46 ± 192,94cm2, néctar (1.219 ± 149,58cm2, mel (563,47 ± 807,93cm2, pólen (139,09 ± 80,30cm2 e percentagem de infestação por V. destructor (10,50 ± 4,30%. As colônias perdidas suplementadas com favos de mel possuíam áreas de ovo-larva (7,92 ± 9,35cm2, pupas (60,71 ± 63,94 cm2, néctar (658,22 ± 1.040,94cm2, mel (122,62 ± 49,45cm2, pólen (53,58 ± 72,39cm2 e percentagem de infestação por V. destructor (14,40 ± 1,56%. As causas das perdas em parte podem ser atribuídas à infestação por V. destructor.

  5. Relacionamento, cooperação e governança em arranjos produtivos locais: o caso do APL de madeira e móveis do estado de Rondônia Relations, cooperation and governance in local production arrangements: the case of APL wooden furniture and state Rondônia

    Directory of Open Access Journals (Sweden)

    Marcia Cristina Teixeira


    Full Text Available Este estudo tem por objetivo identificar os agentes econômicos e institucionais do arranjo produtivo local APL de madeira e móveis do Estado de Rondônia e analisar suas formas de relacionamento, cooperação e governança. Foram utilizados os procedimentos metodológicos relativos ao estudo de caso, com a adoção de uma perspectiva de análise transversal. O nível de análise considerado foi o APL e a unidade de análise foram os gestores tanto das empresas quando das instituições envolvidas. A população abrangida na pesquisa somou trinta e cinco organizações integrantes do arranjo produtivo local de madeira e móveis de Rondônia, das quais vinte e quatro foram visitadas e seus dirigentes, entrevistados. Para a realização da pesquisa, foram utilizados dados primários, coletados por meio de entrevistas semi-estruturadas e observação direta não-participante do pesquisador, além de dados secundários. Os resultados levam a concluir que o APL de madeira e móveis do Estado de Rondônia ainda não existe como um arranjo propriamente dito, mas sim como três subgrupos com estágios diferentes em seus laços de cooperação. A colaboração foi percebida dentro de um dos subgrupos, tornando as empresas participantes mais bem preparadas e capacitadas. Portanto, o desafio é entender esse APL com necessidades e capacidades diferentes, onde as empresas se encontram em diferentes estágios de desenvolvimento.This research had as objective to identify economic and institutional agents of Rondônia wood and furniture cluster and analyze its relationship, cooperation and governance. The methodological approach were the case study, with the adoption of a perspective of transversal analysis. The analysis level was the cluster and the analysis unit were the cluster and institutional management. The population was of thirty-five participant organizations of the wood and furniture cluster of Rondônia and, from those, twenty-four were visited

  6. A sensitive fluorescence reporter for monitoring quorum sensing regulated protease production in Vibrio harveyi. (United States)

    Rajamani, Sathish; Sayre, Richard T


    Many bacteria produce and secrete proteases during host invasion and pathogenesis. Vibrio harveyi, an opportunistic pathogen of shrimp, is known to use a two-component quorum sensing (QS) mechanism for coordination of gene expression including protease secretion at high population densities. We examined the role of V. harveyi's QS signaling molecules, N-(3-hydroxybutanoyl)-L-homoserine lactone (AI-1) and the boron derivative of autoinducer-2 (BAI-2) in extracellular protease production. A fusion protein, M3CLPY (Rajamani et al., 2007), consisting of a large protease sensitive BAI-2 mutant receptor LuxP (~38kDa) flanked by two protease insensitive cyan and yellow variants of GFP (~28kDa each) was utilized as a substrate to detect secreted protease activity. The M3CLPY fusion, with the addition of wild-type V. harveyi (BB120) cell-free culture filtrate showed a time-dependent loss in fluorescence resonance energy transfer (FRET) associated with the cleavage of the LuxP linker protein and hence separation of the two fluorophores. This cleavage of LuxP linker protein leading to decreased FRET efficiency was further confirmed by immunoblotting using anti-GFP antibody. The addition of cell-free filtrates from strains defective in one or both of the two-component QS pathways: luxN(-) (defective in AI-1), luxS(-) (defective in BAI-2), and luxN(-)/luxS(-) (defective in both AI-1/BAI-2) showed differential levels of protease production. The observed protease activities were most pronounced in wild-type, followed by the AI-1 defective mutant (BB170) and the least for luxS(-) mutant (MM30) and luxN(-)/luxS(-) double mutant (MM32) strains. Incidentally, the lowest protease producing strains MM30 and MM32 were both defective in BAI-2 production. This observation was validated by addition of synthetic BAI-2 to MM30 and MM32 strains to restore protease production. Our results indicate that BAI-2 signaling in the two-component QS pathway plays the key role in regulating

  7. Studies on the Catalytic Properties of Partially Purified Alkaline Proteases from Some Selected Microorganisms

    Directory of Open Access Journals (Sweden)

    Titilayo Olufunke Femi-Ola


    Full Text Available Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis.Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that ofLactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis,Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca+2 and Mg+2. Enzyme activities were inhibited strongly by Cu2+ and Hg2+ but were not

  8. Expression profile of the Schistosoma japonicum degradome reveals differential protease expression patterns and potential anti-schistosomal intervention targets.

    Directory of Open Access Journals (Sweden)

    Shuai Liu


    Full Text Available Blood fluke proteases play pivotal roles in the processes of invasion, nutrition acquisition, immune evasion, and other host-parasite interactions. Hundreds of genes encoding putative proteases have been identified in the recently published schistosome genomes. However, the expression profiles of these proteases in Schistosoma species have not yet been systematically analyzed. We retrieved and culled the redundant protease sequences of Schistosoma japonicum, Schistosoma mansoni, Echinococcus multilocularis, and Clonorchis sinensis from public databases utilizing bioinformatic approaches. The degradomes of the four parasitic organisms and Homo sapiens were then comparatively analyzed. A total of 262 S. japonicum protease sequences were obtained and the expression profiles generated using whole-genome microarray. Four main clusters of protease genes with different expression patterns were identified: proteases up-regulated in hepatic schistosomula and adult worms, egg-specific or predominantly expressed proteases, cercaria-specific or predominantly expressed proteases, and constantly expressed proteases. A subset of protease genes with different expression patterns were further validated using real-time quantitative PCR. The present study represents the most comprehensive analysis of a degradome in Schistosoma species to date. These results provide a firm foundation for future research on the specific function(s of individual proteases and may help to refine anti-proteolytic strategies in blood flukes.

  9. Developing HIV-1 Protease Inhibitors through Stereospecific Reactions in Protein Crystals. (United States)

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


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

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

    Directory of Open Access Journals (Sweden)

    Folasade M. Olajuyigbe


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

  11. Structural Insights into the Allosteric Operation of the Lon AAA+ Protease. (United States)

    Lin, Chien-Chu; Su, Shih-Chieh; Su, Ming-Yuan; Liang, Pi-Hui; Feng, Chia-Cheng; Wu, Shih-Hsiung; Chang, Chung-I


    The Lon AAA+ protease (LonA) is an evolutionarily conserved protease that couples the ATPase cycle into motion to drive substrate translocation and degradation. A hallmark feature shared by AAA+ proteases is the stimulation of ATPase activity by substrates. Here we report the structure of LonA bound to three ADPs, revealing the first AAA+ protease assembly where the six protomers are arranged alternately in nucleotide-free and bound states. Nucleotide binding induces large coordinated movements of conserved pore loops from two pairs of three non-adjacent protomers and shuttling of the proteolytic groove between the ATPase site and a previously unknown Arg paddle. Structural and biochemical evidence supports the roles of the substrate-bound proteolytic groove in allosteric stimulation of ATPase activity and the conserved Arg paddle in driving substrate degradation. Altogether, this work provides a molecular framework for understanding how ATP-dependent chemomechanical movements drive allosteric processes for substrate degradation in a major protein-destruction machine. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Baixo peso ao nascer em coorte de recém-nascidos em Goiânia-Brasil no ano de 2000 Low birth weight in a cohort of newborns in Goiânia-Brazil in 2000

    Directory of Open Access Journals (Sweden)

    Margareth Rocha Peixoto Giglio


    Full Text Available OBJETIVO: analisar o peso ao nascer da coorte de recém-nascidos do ano 2000, em Goiânia, pela determinação do coeficiente de mortalidade e probabilidade de sobrevivência neonatal, estratificados por categorias de peso ao nascer e, ainda, pela identificação dos fatores associados ao baixo peso ao nascer (BPN. MÉTODOS: estudo de coorte retrospectivo, constituído por linkage dos arquivos do SIM (Sistema de Informações de Mortalidade e do SINASC (Sistema de Informações de Nascimentos. Foram calculados coeficientes de mortalidade neonatal para as categorias de peso ao nascer e construído um gráfico de probabilidades de sobrevivência neonatal por meio de análise de regressão linear. Foram identificados fatores de risco para o BPN mediante análise univariada (RR e regressão logística, considerando-se nível de significância de 5%. RESULTADOS: a incidência de BPN foi de 6,9%, sendo que 140 (66,8% óbitos neonatais ocorreram nesse grupo. Trinta por cento dos óbitos se deram na categoria de peso entre 1.500-2.500 g. Os fatores identificados como de risco para o BPN foram: prematuridade, presença de malformações congênitas, mães com idade em extremos reprodutivos, residência na região noroeste do município, baixo número de consultas no pré-natal, parto em hospital público e sexo feminino. CONCLUSÃO: a incidência de BPN foi semelhante aos países desenvolvidos e os coeficientes de mortalidade neonatal, por categoria de peso, aquém dos encontrados naqueles países. Os resultados encontrados orientam atenção para: prematuridade, hospitais públicos e região noroeste de Goiânia.PURPOSE: to analyze birth weight in a cohort of newborns for the year 2000, in Goiânia, by determining the coefficient of mortality and neonatal survival probability, stratified by categories of birth weight, and also, through the identification of factors associated with low birth weight (LBW. METHODS: a retrospective cohort study, made

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

    DEFF Research Database (Denmark)

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


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

  14. Targeting cysteine proteases in trypanosomatid disease drug discovery. (United States)

    Ferreira, Leonardo G; Andricopulo, Adriano D


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

  15. Tendência secular de crescimento em escolares de Paulínia, São Paulo-Brasil (1979/80 - 1993/94 Secular trends of growth of schoolchildren from Paulínia, São Paulo - Brazil (1979/80 - 1993/94

    Directory of Open Access Journals (Sweden)

    Denise Barbieri Marmo


    Full Text Available OBJETIVO: Avaliar a tendência secular de altura, peso e da relação peso/altura em escolares de Paulínia, São Paulo, Brasil, entre 1979/80 e 1993/94. MÉTODOS: Medidas antropométricas de peso (P e altura (A e da relação P/A de 1.903 crianças de 6,5 a 12,5 anos (51,5% M e 48,5% F de escolas públicas de Paulínia foram comparadas com dados de estudo anterior realizado na mesma cidade, com intervalo de 15 anos. Calculou-se o incremento por década e alisou-se os dados pela técnica de médias e medianas móveis (3H3H3. RESULTADOS: As médias de altura e peso foram sempre superiores às do estudo anterior, com incrementos positivos. Os incrementos para a altura variaram de 1,13 a 5 cm no sexo masculino e 1,2 a 4,33 cm nas meninas, já para o peso a variação foi de 0,53 a 4,13 kg nos meninos e 0,87 a 3,0 kg nas meninas. Para a relação P/A, as médias dos dois estudos foram muito semelhantes em ambos os sexos. CONCLUSÕES: A tendência secular positiva da estatura e do peso neste período deve-se principalmente ao desenvolvimento econômico, de saneamento básico, de habitação, de educação e na área de saúde ocorrido na cidade de Paulínia. A tendência secular positiva encontrada foi semelhante à observada em países desenvolvidos após a II Guerra Mundial e de outros trabalhos brasileiros.BACKGROUND: The purpose of this study was to evaluate the secular trends in height, weight and weight/height of schoolchildren from the city of Paulínia, São Paulo, Brazil between 1979/80 and 1993/94. METHODS: Anthropometric measurements (height, weight and weight/height of 1,903 children (6.5-12.5y; 51.5% M: 48.5% F, from Paulínia public schools were compared with data from a previous study carried out in the same city 15 years earlier. Decade increments were calculated and data was smoothed by the technique of means and medians (3H3H3. RESULTS: Height and weight mean values were always greater than those of the previous study, with positive

  16. Biochemical Analysis Reveals the Multifactorial Mechanism of Histone H3 Clipping by Chicken Liver Histone H3 Protease

    KAUST Repository

    Chauhan, Sakshi


    Proteolytic clipping of histone H3 has been identified in many organisms. Despite several studies, the mechanism of clipping, the substrate specificity, and the significance of this poorly understood epigenetic mechanism are not clear. We have previously reported histone H3 specific proteolytic clipping and a protein inhibitor in chicken liver. However, the sites of clipping are still not known very well. In this study, we attempt to identify clipping sites in histone H3 and to determine the mechanism of inhibition by stefin B protein, a cysteine protease inhibitor. By employing site-directed mutagenesis and in vitro biochemical assays, we have identified three distinct clipping sites in recombinant human histone H3 and its variants (H3.1, H3.3, and H3t). However, post-translationally modified histones isolated from chicken liver and Saccharomyces cerevisiae wild-type cells showed different clipping patterns. Clipping of histone H3 N-terminal tail at three sites occurs in a sequential manner. We have further observed that clipping sites are regulated by the structure of the N-terminal tail as well as the globular domain of histone H3. We also have identified the QVVAG region of stefin B protein to be very crucial for inhibition of the protease activity. Altogether, our comprehensive biochemical studies have revealed three distinct clipping sites in histone H3 and their regulation by the structure of histone H3, histone modifications marks, and stefin B.

  17. Directed mutagenesis affects recombination in Azospirillum brasilense nif genes

    Directory of Open Access Journals (Sweden)

    C.P. Nunes


    Full Text Available In order to improve the gene transfer/mutagenesis system for Azospirillum brasilense, gene-cartridge mutagenesis was used to replace the nifD gene with the Tn5 kanamycin resistance gene. The construct was transferred to A. brasilense by electrotransformation. Of the 12 colonies isolated using the suicide plasmid pSUP202 as vector, only four did not show vector integration into the chromosome. Nevertheless, all 12 colonies were deficient in acetylene reduction, indicating an Nif- phenotype. Four Nif- mutants were analyzed by Southern blot, using six different probes spanning the nif and Km r genes and the plasmid vector. Apparently, several recombination events occurred in the mutant genomes, probably caused mainly by gene disruption owing to the mutagenesis technique used: resistance gene-cartridge mutagenesis combined with electrotransformation.Com o objetivo de melhorar os sistemas de transferência gênica e mutagênese para Azospirillum brasilense, a técnica de mutagênese através do uso de um gene marcador ("gene-cartridge mutagenesis" foi utilizada para substituir a região genômica de A. brasilense correspondente ao gene nifD por um segmento de DNA do transposon Tn5 contendo o gene que confere resistência ao antibiótico canamicina. A construção foi transferida para a linhagem de A. brasilense por eletrotransformação. Doze colônias transformantes foram isoladas com o plasmídeo suicida pSUP202 servindo como vetor. Dessas, somente quatro não possuíam o vetor integrado no cromossomo da bactéria. Independentemente da integração ou não do vetor, as 12 colônias foram deficientes na redução do gás acetileno, evidenciando o fenótipo Nif -. Quatro mutantes Nif - foram analisados através da técnica de Southern blot, utilizando-se seis diferentes fragmentos contendo genes nif, de resistência à canamicina e do vetor como sondas. Os resultados sugerem a ocorrência de eventos recombinacionais variados no genoma dos mutantes. A

  18. Effects of protease inhibitors on radiation transformation in vitro

    International Nuclear Information System (INIS)

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


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

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

    International Nuclear Information System (INIS)

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


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

  20. Identification of a Degradation Signal Sequence within Substrates of the Mitochondrial i-AAA Protease. (United States)

    Rampello, Anthony J; Glynn, Steven E


    The i-AAA protease is a component of the mitochondrial quality control machinery that regulates respiration, mitochondrial dynamics, and protein import. The protease is required to select specific substrates for degradation from among the diverse complement of proteins present in mitochondria, yet the rules that govern this selection are unclear. Here, we reconstruct the yeast i-AAA protease, Yme1p, to examine the in vitro degradation of two intermembrane space chaperone subunits, Tim9 and Tim10. Yme1p degrades Tim10 more rapidly than Tim9 despite high sequence and structural similarity, and loss of Tim10 is accelerated by the disruption of conserved disulfide bonds within the substrate. An unstructured N-terminal region of Tim10 is necessary and sufficient to target the substrate to the protease through recognition of a short phenylalanine-rich motif, and the presence of similar motifs in other small Tim proteins predicts robust degradation by the protease. Together, these results identify the first specific degron sequence within a native i-AAA protease substrate. Copyright © 2017 Elsevier Ltd. All rights reserved.

  1. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.


    Purpose: To provide a recombiner which is capable of converting hydrogen gas into water by use of high-frequency heating at comparatively low temperatures and is safe and cheap in cost. Constitution: Hydrogen gas is introduced from an outer pipeline to the main structure of a recombiner, and when it passes through the vicinity of the central part of the recombiner, it is reacted with copper oxide (CuO 2 ) heated to a temperature more than 300 0 C by a high-frequency heater, and converted gently into water by reduction operation (2H 2 + CuO 2 → Cu + 2H 2 O). The thus prepared water is exhausted through the outer pipeline to a suppression pool. A part of hydrogen gas which has not been converted completely into water by the reaction and is remaining as hydrogen is recovered through exhaust nozzles and again introduced into the main structure of the recombiner. (Yoshino, Y.)

  2. HIV protease inhibitors in pregnancy : pharmacology and clinical use. (United States)

    Andany, Nisha; Loutfy, Mona R


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

  3. Protease Inhibitors of Parasitic Flukes: Emerging Roles in Parasite Survival and Immune Defence. (United States)

    Ranasinghe, Shiwanthi L; McManus, Donald P


    Protease inhibitors play crucial roles in parasite development and survival, counteracting the potentially damaging immune responses of their vertebrate hosts. However, limited information is currently available on protease inhibitors from schistosomes and food-borne trematodes. Future characterization of these molecules is important not only to expand knowledge on parasitic fluke biology but also to determine whether they represent novel vaccine and/or drug targets. Moreover, protease inhibitors from flukes may represent lead compounds for the development of a new range of therapeutic agents against inflammatory disorders and cancer. This review discusses already identified protease inhibitors of fluke origin, emphasizing their biological function and their possible future development as new intervention targets. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang


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

  5. Coxsackievirus B3 2A protease promotes encephalomyocarditis virus replication. (United States)

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


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

  6. Effect of amino acids on the repression of alkaline protease synthesis in haloalkaliphilic Nocardiopsis dassonvillei

    Directory of Open Access Journals (Sweden)

    Amit K. Sharma


    Full Text Available A newly isolated salt-tolerant alkaliphilic actinomycete, Nocardiopsis dassonvillei strain OK-18 grows on mineral salts medium with glucose as carbon source. It also grows and produces protease with amino acids as sole carbon source. The synthesis of extracellular alkaline protease parallel to growth was repressible by substrate concentrations. The absolute production of the protease was delinked with growth under nutritional stress, as protease production was high, despite poor growth. When amino acids served as the sole source of carbon and nitrogen, the enzyme production was significantly controlled by the number of amino acids. Maximal protease production was achieved with proline, asparagine, tyrosine, alanine, methionine and valine as sole source of carbon and nitrogen in minimal medium. With the increasing number of different amino acids in the presence and absence of glucose, the protease production was synergistically lower as compared to complex medium.

  7. Metabolic complications associated with HIV protease inhibitor therapy. (United States)

    Nolan, David


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



    Duque-Estrada, Megan


    Este trabalho investiga o uso dos marcadores pragmáticos né e sabe em narrativas orais acerca do imaginário da Amazônia, tendo como objeto a identificação da ideia de relevância em histórias de encantamento e assombração. É uma pesquisa de cunho empírico-indutivo, com suporte teórico básico da Análise da Conversação, complementado por conceitos oriundos da Pragmática, do Princípio da Pertinência e das Estruturas de Expectativa, além de estudos sobre a narrativa oral, na linha da Sociolinguíst...

  9. DURAMAZ, um Sistema de Indicadores de Desenvolvimento Sustentável na Amazônia

    Directory of Open Access Journals (Sweden)

    Martine Droulers


    Full Text Available A análise do processo de sustentabilidade representa um grande desafio científico enquanto a Amazônia constitui um verdadeiro laboratório onde estão sendo testadas inúmeras concepções desse conceito. Haveria pontos ou mecanismos comuns entre estas? Esse é o ponto que pesquisou o programa DURAMAZ, que teve como objetivo de desvendar fatores chaves, ou “determinantes” do desenvolvimento sustentável (DS na Amazô