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Sample records for active recombinant cathepsin

  1. Expression and Purification of Active Recombinant Cathepsin C (Dipeptidyl Aminopeptidase I of Kuruma Prawn Marsupenaeus japonicus in Insect Cells

    Directory of Open Access Journals (Sweden)

    Gao-Feng Qiu

    2009-01-01

    Full Text Available Cathepsin C (CTSC is a lysosomal cysteine protease belonging to the papain superfamily. Our previous study showed that CTSC precursor (zymogen is localized exclusively in cortical rods (CRs of mature oocyte in the kuruma prawn Marsupenaeus japonicus, suggesting that CTSC might have roles on regulating release and/or formation of a jelly layer. In this study, enzymically active CTSC of the kuruma prawn was prepared by recombinant expression in the High Five insect cell line. The recombinant enzyme with a polyhistidine tag at its C-terminus was considered to be initially secreted into the culture medium as an inactive form of zymogen, because Western blot with anti-CTSC antibody detected a 51 kDa protein corresponding to CTSC precursor. After purification by affinity chromatography on nickel-iminodiacetic acid resin, the enzyme displayed three forms of 51, 31, and 30 kDa polypeptides. All of the forms can be recognized by antiserum raised against C-terminal polyhistidine tag, indicating that the 31 and 30 kDa forms were generated from 51 kDa polypeptide by removal of a portion of the N-terminus of propeptide. Following activation at pH 5.5 and 37∘C for 40 hours under native conditions, the recombinant CTSC (rCTSC exhibited increased activity against the synthetic substrate Gly-Phe-β-naphthylamide and optimal pH at around 5. The purified rCTSC will be useful for further characterization of its exact physiological role on CRs release and/or formation of a jelly layer in kuruma prawn.

  2. Vaccine potential of recombinant cathepsin B against Fasciola gigantica.

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    Chantree, Pathanin; Phatsara, Manussabhorn; Meemon, Krai; Chaichanasak, Pannigan; Changklungmoa, Narin; Kueakhai, Pornanan; Lorsuwannarat, Natcha; Sangpairoj, Kant; Songkoomkrong, Sineenart; Wanichanon, Chaitip; Itagaki, Tadashi; Sobhon, Prasert

    2013-09-01

    In Fasciola gigantica, cathepsin Bs, especially cathepsin B2 and B3 are expressed in early juvenile stages, and are proposed to mediate the invasion of host tissues. Thus they are thought to be the target vaccine candidates that can block the invasion and migration of the juvenile parasite. To evaluate their vaccine potential, the recombinant cathepsin B2 (rFgCatB2) and cathepsin B3 (rFgCatB3) were expressed in yeast, Pichia pastoris, and used to immunize mice in combination with Freund's adjuvant to evaluate the protection against the infection by F. gigantica metacercariae, and the induction of immune responses. Mice immunized with both recombinant proteins exhibited high percent of parasite reduction at 60% for rFgCatB2 and 66% for rFgCatB3. Immunization by both antigens induced continuously increasing levels of IgG1 and IgG2a with a higher level of IgG1 isotype, indicating the mixed Th1/Th2 responses with Th2 predominating. When examined individually, the higher levels of IgG1 and IgG2a were correlated with the lower numbers of worm recoveries. Thus, both cathepsin B2 and cathepsin B3 are plausible vaccine candidates whose potential should be further tested in large economic animals. Copyright © 2013 Elsevier Inc. All rights reserved.

  3. Multiplex Cathepsin Zymography to Detect Amounts of Active Cathepsins K, L, S, and V.

    Science.gov (United States)

    Platt, Manu O

    2017-01-01

    Cysteine cathepsins are powerful proteases that can degrade other proteins, among which are the extracellular matrix proteins collagen and elastin. Multiplex cathepsin zymography is an assay that can quantify the amount of active cathepsins in a cell or tissue preparation. This method works for measuring the amounts of active cathepsins K, L, S, and V in a cell or tissue preparation without requiring the use of antibodies for specific identification which tremendously reduces cost. This chapter will demonstrate the utility and interpretation of this method with mammalian cells and tissue to quantify amounts of active cathepsins K, L, S, and V without complicating signals of the procathepsin. Multiplex cathepsin zymography has many advantages: (1) it separates cathepsins K, L, S, and V by electrophoretic migration distance, (2) allows visual confirmation of cathepsin identity, (3) does not detect procathepsins, and (4) can be quantified with densitometry.

  4. Monoclonal antibody against recombinant Fasciola gigantica cathepsin L1H could detect juvenile and adult cathepsin Ls of Fasciola gigantica.

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    Wongwairot, Sirima; Kueakhai, Pornanan; Changklungmoa, Narin; Jaikua, Wipaphorn; Sansri, Veerawat; Meemon, Krai; Songkoomkrong, Sineenart; Riengrojpitak, Suda; Sobhon, Prasert

    2015-01-01

    Cathepsin Ls (CatLs), the major cysteine protease secreted by Fasciola spp., are important for parasite digestion and tissue invasion. Fasciola gigantica cathepsin L1H (FgCatL1H) is the isotype expressed in the early stages for migration and invasion. In the present study, a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1H (rFgCatL1H) was produced by hybridoma technique using spleen cells from BALB/c mice immunized with recombinant proFgCatL1H (rproFgCatL1H). This MoAb is an immunoglobulin (Ig)G1 with κ light chain isotype. The MoAb reacted specifically with rproFgCatL1H, the native FgCatL1H at a molecular weight (MW) 38 to 48 kDa in the extract of whole body (WB) of metacercariae and newly excysted juvenile (NEJ) and cross-reacted with rFgCatL1 and native FgCatLs at MW 25 to 28 kDa in WB of 2- and 4-week-old juveniles, adult, and adult excretory-secretory (ES) fractions by immunoblotting and indirect ELISA. It did not cross-react with antigens in WB fractions from other parasites, including Gigantocotyle explanatum, Paramphistomum cervi, Gastrothylax crumenifer, Eurytrema pancreaticum, Setaria labiato-papillosa, and Fischoederius cobboldi. By immunolocalization, MoAb against rFgCatL1H reacted with the native protein in the gut of metacercariae and NEJ and also cross-reacted with CatL1 in 2- and 4-week-old juveniles and adult F. gigantica. Therefore, FgCatL1H and its MoAb may be used for immunodiagnosis of both early and late fasciolosis in ruminants and humans.

  5. Profilin 1 as a Target for Cathepsin X Activity in Tumor Cells

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    Pečar Fonović, Urša; Jevnikar, Zala; Rojnik, Matija; Doljak, Bojan; Fonović, Marko; Jamnik, Polona; Kos, Janko

    2013-01-01

    Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies. PMID:23326535

  6. Profilin 1 as a target for cathepsin X activity in tumor cells.

    Directory of Open Access Journals (Sweden)

    Urša Pečar Fonović

    Full Text Available Cathepsin X has been reported to be a tumor promotion factor in various types of cancer; however, the molecular mechanisms linking its activity with malignant processes are not understood. Here we present profilin 1, a known tumor suppressor, as a target for cathepsin X carboxypeptidase activity in prostate cancer PC-3 cells. Profilin 1 co-localizes strongly with cathepsin X intracellularly in the perinuclear area as well as at the plasma membrane. Selective cleavage of C-terminal amino acids was demonstrated on a synthetic octapeptide representing the profilin C-terminal region, and on recombinant profilin 1. Further, intact profilin 1 binds its poly-L-proline ligand clathrin significantly better than it does the truncated one, as shown using cathepsin X specific inhibitor AMS-36 and immunoprecipitation of the profilin 1/clathrin complex. Moreover, the polymerization of actin, which depends also on the binding of poly-L-proline ligands to profilin 1, was promoted by AMS-36 treatment of cells and by siRNA cathepsin X silencing. Our results demonstrate that increased adhesion, migration and invasiveness of tumor cells depend on the inactivation of the tumor suppressive function of profilin 1 by cathepsin X. The latter is thus designated as a target for development of new antitumor strategies.

  7. Expression, purification and auto-activation of cathepsin E from insect cells.

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    Železnik, Tajana Z; Puizdar, Vida; Dolenc, Iztok

    2015-01-01

    Cathepsin E is an aspartic protease that belongs to the pepsin family. This protease is similar to cathepsin D but differs in its tissue distribution and cell localization. Elevated levels of this enzyme are linked to several tumors, including devastating pancreatic ductal adenocarcinoma. In this manuscript, we present a new protocol for the high-yield purification of recombinant human cathepsin E in the baculovirus expression system. The recombinant protein was produced by the Sf9 insect cell line and secreted into the medium in the form of an inactive zymogen. Procathepsin E was purified using ion-exchange and size exclusion chromatographies followed by pepstatin- and heparin-affinity chromatography steps. The zymogen was activated at an acidic pH, resulting in a high yield of the activated intermediate of cathepsin E. The enzymatic activity, stability, and molecular weight corresponded to those of cathepsin E. The new purification procedure will promote further studies of this enzyme to improve the understanding of its structure-function relationship and consequently enable the development of better therapeutic approaches.

  8. Protection against Fasciola gigantica infection in mice by vaccination with recombinant juvenile-specific cathepsin L.

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    Sansri, Veerawat; Meemon, Krai; Changklungmoa, Narin; Kueakhai, Pornanan; Chantree, Pathanin; Chaichanasak, Pannigan; Lorsuwannarat, Natcha; Itagaki, Tadashi; Sobhon, Prasert

    2015-03-24

    Fasciola gigantica cathepsin L1H (FgCatL1H) is one of the major cathepsin L released by juveniles of F. gigantica to aid in the invasion of host's tissues. Due to its high sequence similarity with other cathepsin L (CatL) isoforms of late stage F. gigantica, it was considered to be a good vaccine candidate that can block all CatL-mediated protease activities and affect juveniles as well as adult parasites. In this study, recombinant proFgCatL1H protein expressed in yeast, Pichia pastoris, system was mixed with Freund's adjuvants and used to subcutaneously immunize mice that were later challenged with metacercariae of F. gigantica. The percentage of worm protection in the rproFgCatL1H-vaccinated mice compared to the non-immunized and adjuvant control mice were approximately 62.7% and 66.1%, respectively. Anti-rproFgCatL1H antisera collected from vaccinated mice reacted specifically with rproFgCatL1H and other cathepsin L isoforms of F. gigantica, but the antibodies did not cross react with antigens from other trematode and nematode parasites, including Eurytrema pancreaticum, Opisthorchis viverrini, Fischoederius cobboldi, Cotylophoron cotylophorum, Gigantocotyle explanatum, Paramphistomum cervi, and Setaria labiato-papillosa. The levels of IgG1 and IgG2a in mouse sera increased significantly at two weeks after immunization and were highest during the sixth to eighth weeks after immunization. The IgG1 level was higher than IgG2a at all periods of immunization, implicating the dominance of the Th2 response. The levels of IgG1 and IgG2a in the immune sera were shown to be strongly correlated with the numbers of worm recovery, and the correlation coefficient was higher for IgG1. The levels of serum aspartate aminotransferase and alanine transaminase were significantly lower in the sera of rproFgCatL1H-vaccinated mice than in the infected control mice indicating a lower degree of liver damage. This study demonstrated a high potential of FgCatL1H vaccine, and its

  9. Follicular thyroglobulin induces cathepsin H expression and activity in thyrocytes

    International Nuclear Information System (INIS)

    Oda, Kenzaburo; Luo, Yuqian; Yoshihara, Aya; Ishido, Yuko; Sekihata, Kengo

    2017-01-01

    Thyroglobulin (Tg) stored in thyroid follicles exerts a potent negative-feedback effect on each step of pre-hormone biosynthesis, including Tg gene transcription and iodine uptake and organification, by suppressing the expression of specific transcription factors that regulate these steps. Pre-hormones are stored in the follicular colloid before being reabsorbed. Following lysosomal proteolysis of its precursor, thyroid hormone (TH) is released from thyroid follicles. Although the suppressive effects of follicular Tg on each step of pre-hormone biosynthesis have been extensively characterized, whether follicular Tg accumulation also affects hormone reabsorption, proteolysis, and secretion is unclear. In this study we explored whether follicular Tg can regulate the expression and function of the lysosomal endopeptidases cathepsins. We found that in the rat thyroid cell line FRTL-5 follicular Tg induced cathepsin H mRNA and protein expression, as well as cathepsin H enzyme activity. Double immunofluorescence staining showed that Tg endocytosis promoted cathepsin H translocalization into lysosomes where it co-localized with internalized Tg. These results suggest that cathepsin H is an active participant in lysosome-mediated pre-hormone degradation, and that follicular Tg stimulates mobilization of pre-hormones by activating cathepsin H-associated proteolysis pathways. - Highlights: • Follicular Tg increases cathepsin H mRNA and protein levels in rat thyroid cells. • Follicular Tg increases cathepsin H enzyme activity in rat thyroid cells. • After Tg stimulation cathepsin H co-localizes to lysosomes with follicular Tg. • Cathepsin H promotes hormone secretion by lysosome-mediated mechanisms.

  10. Cysteine Cathepsins in the secretory vesicle produce active peptides: Cathepsin L generates peptide neurotransmitters and cathepsin B produces beta-amyloid of Alzheimer's disease.

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    Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory

    2012-01-01

    Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.

  11. Complex modulation of peptidolytic activity of cathepsin D by sphingolipids

    Czech Academy of Sciences Publication Activity Database

    Žebrakovská, Iva; Máša, Martin; Srp, Jaroslav; Horn, Martin; Vávrová, K.; Mareš, Michael

    2011-01-01

    Roč. 1811, č. 12 (2011), s. 1097-1104 ISSN 1388-1981 R&D Projects: GA AV ČR IAA400550705 Institutional research plan: CEZ:AV0Z40550506 Keywords : sphingolipid * phospholipid * inhibition * activation * cathepsin D * enzyme regulation Subject RIV: CE - Biochemistry Impact factor: 5.269, year: 2011

  12. [Effect of cryoconservation of platelets on polyamine level and cathepsin D activity].

    Science.gov (United States)

    Kovtunova, M E; Selezneva, O M; Vorozhtsova, S I; Kunof, V K

    1992-01-01

    Alterations in activity of cathepsin D as well as in content of polyamines spermine, spermidine, x-fraction (apparently, acetyl spermidine) and putrescine were studied in blood platelets depending on conditions of conservation. The enzyme activity and content of polyamines correlated directly with duration of the cell concentrates storage. Cathepsin D and polyamines appear to be involved in responses to stress adaptation.

  13. The development and characterization of an ELISA specifically detecting the active form of cathepsin K

    DEFF Research Database (Denmark)

    Sun, S; Karsdal, M A; Bay-Jensen, A C

    2013-01-01

    Cathepsin K plays essential roles in bone resorption and is intensely investigated as a therapeutic target for the treatment of osteoporosis. Hence an assessment of the active form of cathepsin K may provide important biological information in metabolic bone diseases, such as osteoporosis or anky...

  14. Distribution of Cathepsin D Activity between Lysosomes and a Soluble Fraction of Marinating Brine.

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    Szymczak, Mariusz

    2016-08-01

    This paper is the first ever to describe the phenomenon of bimodal distribution of cathepsin D in the lysosomal and soluble fractions of brine left after herring marinating. Up to 2 times higher cathepsin D activity was observed in the lysosome fraction. Activity of cathepsin D in brine increased according to the logarithmic function during low frequency-high power ultrasounds treatment or according to the linear function after multiple freezing-thawing of brine. Activity enhancement was achieved only in the brine devoid of lipids and suspension. Study results show also that measurement of lysosomal cathepsin D activity in the marinating brine requires also determining cathepsin E activity. Decreasing pore size of microfilter from 2.7 to 0.3 μm significantly reduced the lysosome content in the brine. The presence of lysosomes and the possibility of their separation as well as the likely release of cathepsins shall be considered during industrial application of the marinating brine, as new cathepsins preparations in fish and meat technology. © 2016 Institute of Food Technologists®

  15. Production and characterization of a monoclonal antibody against recombinant cathepsin L1 of Fasciola gigantica.

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    Anuracpreeda, Panat; Srirakam, Thippawan; Pandonlan, Sudarat; Changklungmoa, Narin; Chotwiwatthanakun, Charoonroj; Tinikul, Yotsawan; Poljaroen, Jaruwan; Meemon, Krai; Sobhon, Prasert

    2014-08-01

    Monoclonal antibodies (MoAbs) against a recombinant cathepsin L1 of Fasciola gigantica (rFgCatL1) were produced in vitro by fusion of BALB/c mice spleen cells immunized with rFgCatL1 and mouse myeloma cells. Reactivity and specificity of these MoAbs were evaluated by indirect ELISA and immunoblotting techniques. Seven MoAb clones were selected from the stable hybridoma clones, namely 1E10, 1F5, 3D11, 4B10, 4D3, 4E3 and 5E7. Clones 1E10, 1F5 and 3D11 were IgM, whereas clones 4B10, 4D3, 4E3 and 5E7 were IgG1. All MoAbs had kappa light chain isotypes. All MoAbs reacted with rCatL1 at molecular weight (MW) 30kDa and with the native CatL1 at MW 27kDa in whole body (WB) extracts of metacercariae (Met), newly excysted juveniles (NEJ), 1, 3, 5-week-old juveniles (Ju), adult WB and adult excretory-secretory (ES) fractions, but not with adult tegumental antigens (TA). All of these MoAbs showed no cross-reactions with antigens of other parasites commonly found in ruminants and human, including Paramphistomum cervi, Eurytrema pancreaticum, Gigantocotyle explanatum, Schistosoma spindale, Schistosoma mansoni, Moniezia benedeni, Avitellina centripunctata, Trichuris sp., Haemonchus placei and Setaria labiato-papillosa. Localization of CatL1 in each developmental stages of F. gigantica by immunoperoxidase technique, using these MoAbs as probes, indicated that CatL1 was present at high concentration in the caecal epithelium and caecal lumen of metacercariae, NEJ, 1, 3, 5-week-old juveniles and adult fluke. This finding indicated that CatL1 is a copiously expressed parasite protein that is released into the ES, thus CatL1 and its MoAb could be a good candidate for immunodiagnosis of fasciolosis in ruminant and human. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. [Changes in active cysteine cathepsins in lysosomes from tissues thyroid papillary carcinomas with various biological characteristics].

    Science.gov (United States)

    Kalinichenko, O V; Myshunina, T M; Tron'ko, M D

    2013-01-01

    To clarify possible role of cysteine cathepsin H, B and L in the proteolytic processes that contribute to the progression of tumor growth in the thyroid, we studied their activity in lysosomes isolated from the tissue of papillary carcinomas. It was shown that for these enzymes there is a dependence of the changes in their activity on a number of biological characteristics of the tumors. Thus, the sharp increase in the activity ofcathepsin H observed in lysosomes of tissue carcinomas category T2 and T3, with intra-and ekstrathyroid and lymphatic invasion of tumor cells. An increase in the activity of cathepsin B is set in the lysosomes of tissue heterogeneous follicular structure, especially in the presence of solid areas, in comparison with typical papillary tumors and in the lysosomes of tissue carcinomas in intrathyroid and cathepsin L-at extrathyroid invasion. A common feature of the enzymes is to increase the activity of cathepsins in lysosomes of tissue nonencapsulated papillary carcinomas. These enzymes probably do not take part in the invasion of tumor cells into blood vessels and in the mechanisms of tumor metastasis to regional lymph nodes. The latter shows no changes in the activity of cathepsins in lysosomes of tissue carcinomas category N1. The results indicate the different role of cathepsin H, B and L in thyroid carcinogenesis, where each enzyme has its specific function.

  17. Activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1999-08-10

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  18. Activation of lysosomal cathepsins in pregnant bovine leukocytes.

    Science.gov (United States)

    Talukder, Md Abdus Shabur; Balboula, Ahmed Zaky; Shirozu, Takahiro; Kim, Sung Woo; Kunii, Hiroki; Suzuki, Toshiyuki; Ito, Tsukino; Kimura, Koji; Takahashi, Masashi

    2018-06-01

    In ruminants, interferon-tau (IFNT) - mediated expression of interferon-stimulated genes in peripheral blood leukocytes (PBLs) can indicate pregnancy. Recently, type 1 IFN-mediated activation of lysosomes and lysosomal cathepsins (CTSs) was observed in immune cells. This study investigated the status of lysosomal CTSs and lysosomes in PBLs collected from pregnant (P) and non-pregnant (NP) dairy cows, and conducted in vitro IFNT stimulation of NP blood leukocytes. Blood samples were collected 0, 7, 14 and 18 days post-artificial insemination, and the peripheral blood mononuclear cells (PBMCs) and polymorphonuclear granulocytes (PMNs) separated. The fluorescent activity of CTSB and CTSK in PMNs significantly increased with the progress of pregnancy, especially on day 18. In vitro supplementation of IFNT significantly increased the activities of CTSB and CTSK in NP PBMCs and PMNs. CTSB expression was significantly higher in PBMCs and PMNs collected from P day-18 cows than from NP cows, whereas there was no difference in CTSK expression. IFNT increased CTSB expression but did not affect CTSK expression. Immunodetection showed an increase of CTSB in P day-18 PBMCs and PMNs. In vitro stimulation of IFNT increased CTSB in NP PBMCs and PMNs. Lysosomal acidification showed a significant increase in P day-18 PBMCs and PMNs. IFNT also stimulated lysosomal acidification. Expressions of lysosome-associated membrane protein (LAMP) 1 and LAMP2 were significantly higher in P day-18 PBMCs and PMNs. The results suggest that pregnancy-specific activation of lysosomal functions by CTS activation in blood leukocytes is highly associated with IFNT during maternal and fetal recognition of pregnancy. © 2018 Society for Reproduction and Fertility.

  19. Active subsite properties, subsite residues and targeting to lysosomes or midgut lumen of cathepsins L from the beetle Tenebrio molitor.

    Science.gov (United States)

    Damasceno, Ticiane F; Dias, Renata O; de Oliveira, Juliana R; Salinas, Roberto K; Juliano, Maria A; Ferreira, Clelia; Terra, Walter R

    2017-10-01

    Cathepsins L are the major digestive peptidases in the beetle Tenebrio molitor. Two digestive cathepsins L (TmCAL2 and TmCAL3) from it had their 3D structures solved. The aim of this paper was to study in details TmCAL3 specificity and properties and relate them to its 3D structure. Recombinant TmCAL3 was assayed with 64 oligopeptides with different amino acid replacements in positions P2, P1, P1' and P2'. Results showed that TmCAL3 S2 specificity differs from the human enzyme and that its specificities also explain why on autoactivation two propeptide residues remain in the enzyme. Data on free energy of binding and of activation showed that S1 and S2' are mainly involved in substrate binding, S1' acts in substrate binding and catalysis, whereas S2 is implied mainly in catalysis. Enzyme subsite residues were identified by docking with the same oligopeptide used for kinetics. The subsite hydrophobicities were calculated from the efficiency of hydrolysis of different amino acid replacements in the peptide and from docking data. The results were closer for S1 and S2' than for S1' and S2, indicating that the residue subsites that were more involved in transition state binding are different from those binding the substrate seen in docking. Besides TmCAL1-3, there are nine other cathepsins L, most of them more expressed at midgut. They are supposed to be directed to lysosomes by a Drosophila-like Lerp receptor and/or motifs in their prodomains. The mannose 6-phosphate lysosomal sorting machinery is absent from T. molitor transcriptome. Cathepsin L direction to midgut contents seems to depend on overexpression. Copyright © 2017 Elsevier Ltd. All rights reserved.

  20. Cathepsin K expression and activity in canine osteosarcoma.

    Science.gov (United States)

    Schmit, J M; Pondenis, H C; Barger, A M; Borst, L B; Garrett, L D; Wypij, J M; Neumann, Z L; Fan, T M

    2012-01-01

    Cathepsin K (CatK) is a lysosomal protease with collagenolytic activity, and its secretion by osteoclasts is responsible for degrading organic bone matrix. People with pathologic bone resorption have higher circulating CatK concentrations. Canine osteosarcoma (OS) cells will possess CatK, and its secretion will be cytokine inducible. Circulating CatK concentrations will be increased in dogs with OS, and will be a surrogate marker of bone resorption. Fifty-one dogs with appendicular OS and 18 age- and weight-matched healthy control dogs. In a prospective study, expressions of CatK mRNA and protein were investigated in OS cells. The inducible secretion and proteolytic activity of CatK from OS cells was assessed in vitro. Serum CatK concentrations were quantified in normal dogs and dogs with OS and its utility as a bone resorption marker was evaluated in dogs with OS treated with palliative radiation and antiresorptive agents. Canine OS cells contain preformed CatK within cytoplasmic vesicles. In OS cells, TGFβ1 induced the secretion of CatK, which degraded bone-derived type I collagen in vitro. CatK concentrations were higher in dogs with OS than healthy dogs (11.3 ± 5.2 pmol/L versus 8.1 ± 5.0 pmol/L, P = .03). In a subset of dogs with OS, pretreatment CatK concentrations gradually decreased after palliative radiation and antiresorptive treatment, from 9.3 ± 3.2 pmol/L to 5.0 ± 3.1 pmol/L, P = .03. Canine OS is associated with pathologic bone resorption, and CatK inhibitors might aid in the management of canine OS-related malignant osteolysis. Copyright © 2011 by the American College of Veterinary Internal Medicine.

  1. Cathepsin K in Lymphangioleiomyomatosis: LAM Cell-Fibroblast Interactions Enhance Protease Activity by Extracellular Acidification.

    Science.gov (United States)

    Dongre, Arundhati; Clements, Debbie; Fisher, Andrew J; Johnson, Simon R

    2017-08-01

    Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells and fibroblasts form lung nodules and it is hypothesized that LAM nodule-derived proteases cause cyst formation and tissue damage. On protease gene expression profiling in whole lung tissue, cathepsin K gene expression was 40-fold overexpressed in LAM compared with control lung tissue (P ≤ 0.0001). Immunohistochemistry confirmed cathepsin K protein was expressed in LAM but not control lungs. Cathepsin K gene expression and protein and protease activity were detected in LAM-associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immunoreactivity predominantly co-localized with LAM-associated fibroblasts. In vitro, fibroblast extracellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced at pH 7 and 6. 621-101 cells reduced extracellular pH with acidification dependent on 621-101 mechanistic target of rapamycin activity and net hydrogen ion exporters, particularly sodium bicarbonate co-transporters and carbonic anhydrases, which were also expressed in LAM lung tissue. In LAM cell-fibroblast co-cultures, acidification paralleled cathepsin K activity, and both were reduced by sodium bicarbonate co-transporter (P ≤ 0.0001) and carbonic anhydrase inhibitors (P = 0.0021). Our findings suggest that cathepsin K activity is dependent on LAM cell-fibroblast interactions, and inhibitors of extracellular acidification may be potential therapies for LAM. Copyright © 2017 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  2. Lysosomal enzyme cathepsin B enhances the aggregate forming activity of exogenous α-synuclein fibrils.

    Science.gov (United States)

    Tsujimura, Atsushi; Taguchi, Katsutoshi; Watanabe, Yoshihisa; Tatebe, Harutsugu; Tokuda, Takahiko; Mizuno, Toshiki; Tanaka, Masaki

    2015-01-01

    The formation of intracellular aggregates containing α-synuclein (α-Syn) is one of the key steps in the progression of Parkinson's disease and dementia with Lewy bodies. Recently, it was reported that pathological α-Syn fibrils can undergo cell-to-cell transmission and form Lewy body-like aggregates. However, little is known about how they form α-Syn aggregates from fibril seeds. Here, we developed an assay to study the process of aggregate formation using fluorescent protein-tagged α-Syn-expressing cells and examined the aggregate forming activity of exogenous α-Syn fibrils. α-Syn fibril-induced formation of intracellular aggregates was suppressed by a cathepsin B specific inhibitor, but not by a cathepsin D inhibitor. α-Syn fibrils pretreated with cathepsin B in vitro enhanced seeding activity in cells. Knockdown of cathepsin B also reduced fibril-induced aggregate formation. Moreover, using LAMP-1 immunocytochemistry and live-cell imaging, we observed that these aggregates initially occurred in the lysosome. They then rapidly grew larger and moved outside the boundary of the lysosome within one day. These results suggest that the lysosomal protease cathepsin B is involved in triggering intracellular aggregate formation by α-Syn fibrils. Copyright © 2015. Published by Elsevier Inc.

  3. Matrix metalloproteinases (MMP) and cathepsin K contribute differently to osteoclastic activities

    DEFF Research Database (Denmark)

    Delaissé, Jean-Marie; Andersen, Thomas L; Engsig, Michael T

    2003-01-01

    The best established proteolytic event of osteoclasts is bone matrix solubilization by the cysteine proteinase cathepsin K. Here, however, we draw the attention on osteoclastic activities depending on matrix metalloproteinases (MMPs). We discuss the observations supporting that MMPs contribute...... significantly to bone matrix solubilization in specific areas of the skeleton and in some developmental and pathological situations. Our discussion takes into account (1) the characteristics of the bone remodeling persisting in the absence of cathepsin K, (2) the ultrastructure of the resorption zone...... in response to inactivation of MMPs and of cathepsin K in different bone types, (3) bone resorption levels in MMP knockout mice compared to wild-type mice, (4) the identification of MMPs in osteoclasts and surrounding cells, and (5) the effect of different bone pathologies on the serum concentrations...

  4. Live imaging of cysteine-cathepsin activity reveals dynamics of focal inflammation, angiogenesis, and polyp growth.

    Directory of Open Access Journals (Sweden)

    Elias Gounaris

    2008-08-01

    Full Text Available It has been estimated that up to 30% of detectable polyps in patients regress spontaneously. One major challenge in the evaluation of effective therapy of cancer is the readout for tumor regression and favorable biological response to therapy. Inducible near infra-red (NIR fluorescent probes were utilized to visualize intestinal polyps of mice hemizygous for a novel truncation of the Adenomatous Polyposis coli (APC gene. Laser Scanning Confocal Microscopy in live mice allowed visualization of cathepsin activity in richly vascularized benign dysplastic lesions. Using biotinylated suicide inhibitors we quantified increased activities of the Cathepsin B & Z in the polyps. More than (3/4 of the probe signal was localized in CD11b(+Gr1(+ myeloid derived suppressor cells (MDSC and CD11b(+F4/80(+ macrophages infiltrating the lesions. Polyposis was attenuated through genetic ablation of cathepsin B, and suppressed by neutralization of TNFalpha in mice. In both cases, diminished probe signal was accounted for by loss of MDSC. Thus, in vivo NIR imaging of focal cathepsin activity reveals inflammatory reactions etiologically linked with cancer progression and is a suitable approach for monitoring response to therapy.

  5. Comparative assessment of ELISAs using recombinant saposin-like protein 2 and recombinant cathepsin L-1 from Fasciola hepatica for the serodiagnosis of human Fasciolosis.

    Directory of Open Access Journals (Sweden)

    Bruno Gottstein

    2014-06-01

    Full Text Available Two recombinant Fasciola hepatica antigens, saposin-like protein-2 (recSAP2 and cathepsin L-1 (recCL1, were assessed individually and in combination in enzyme-linked immunosorbent assays (ELISA for the specific serodiagnosis of human fasciolosis in areas of low endemicity as encountered in Central Europe. Antibody detection was conducted using ProteinA/ProteinG (PAG conjugated to alkaline phosphatase. Test characteristics as well as agreement with results from an ELISA using excretory-secretory products (FhES from adult stage liver flukes was assessed by receiver operator characteristic (ROC analysis, specificity, sensitivity, Youdens J and overall accuracy. Cross-reactivity was assessed using three different groups of serum samples from healthy individuals (n=20, patients with other parasitic infections (n=87 and patients with malignancies (n=121. The best combined diagnostic results for recombinant antigens were obtained using the recSAP2-ELISA (87% sensitivity, 99% specificity and 97% overall accuracy employing the threshold (cut-off to discriminate between positive and negative reactions that maximized Youdens J. The findings showed that recSAP2-ELISA can be used for the routine serodiagnosis of chronic fasciolosis in clinical laboratories; the use of the PAG-conjugate offers the opportunity to employ, for example, rabbit hyperimmune serum for the standardization of positive controls.

  6. Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion.

    Science.gov (United States)

    de Mingo Pulido, Álvaro; de Gregorio, Estefanía; Chandra, Shilpi; Colell, Anna; Morales, Albert; Kronenberg, Mitchell; Marí, Montserrat

    2018-01-01

    Natural killer T (NKT) cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ), and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB) and cathepsin S (CTSS), regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs), probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro .

  7. Differential Role of Cathepsins S and B In Hepatic APC-Mediated NKT Cell Activation and Cytokine Secretion

    Directory of Open Access Journals (Sweden)

    Álvaro de Mingo Pulido

    2018-02-01

    Full Text Available Natural killer T (NKT cells exhibit a specific tissue distribution, displaying the liver the highest NKT/conventional T cell ratio. Upon antigen stimulation, NKT cells secrete Th1 cytokines, including interferon γ (IFNγ, and Th2 cytokines, including IL-4 that recruit and activate other innate immune cells to exacerbate inflammatory responses in the liver. Cysteine cathepsins control hepatic inflammation by regulating κB-dependent gene expression. However, the contribution of cysteine cathepsins other than Cathepsin S to NKT cell activation has remained largely unexplored. Here we report that cysteine cathepsins, cathepsin B (CTSB and cathepsin S (CTSS, regulate different aspects of NKT cell activation. Inhibition of CTSB or CTSS reduced hepatic NKT cell expansion in a mouse model after LPS challenge. By contrast, only CTSS inhibition reduced IFNγ and IL-4 secretion after in vivo α-GalCer administration. Accordingly, in vitro studies reveal that only CTSS was able to control α-GalCer-dependent loading in antigen-presenting cells (APCs, probably due to altered endolysosomal protein degradation. In summary, our study discloses the participation of cysteine cathepsins, CTSB and CTSS, in the activation of NKT cells in vivo and in vitro.

  8. Treatment with a human recombinant monoclonal IgG antibody against oxidized LDL in atherosclerosis-prone pigs reduces cathepsin S in coronary lesions

    DEFF Research Database (Denmark)

    Poulsen, Christian Bo; Al-Mashhadi, Ahmed Ludvigsen; von Wachenfeldt, Karin

    2016-01-01

    and results Thirty-eight hypercholesterolemic minipigs with defective LDL receptors were injected with an oxLDL antibody or placebo weekly for 12 weeks. An 18F-fluorodeoxyglucose positron emission tomography (FDG PET) scan (n = 9) was performed before inclusion and after 3 months of treatment. Blood samples....... There was no effect of treatment on plasma lipid profile, vascular FDG-PET signal or the amount of atherosclerosis in any of the examined arteries. However, immunostaining of coronary lesions revealed reduced cathepsin S positivity in the treated group compared with placebo (4.8% versus 8.2% of intima area, p = 0.......03) with no difference in CD68 or CD163 positivity. Conclusions In hypercholesterolemic minipigs, treatment with a human recombinant monoclonal antibody against oxLDL reduced cathepsin S in coronary lesions without any effect on the burden of atherosclerosis or aortic FDG-PET signal....

  9. Altered Ca2+ homeostasis induces Calpain-Cathepsin axis activation in sporadic Creutzfeldt-Jakob disease.

    Science.gov (United States)

    Llorens, Franc; Thüne, Katrin; Sikorska, Beata; Schmitz, Matthias; Tahir, Waqas; Fernández-Borges, Natalia; Cramm, Maria; Gotzmann, Nadine; Carmona, Margarita; Streichenberger, Nathalie; Michel, Uwe; Zafar, Saima; Schuetz, Anna-Lena; Rajput, Ashish; Andréoletti, Olivier; Bonn, Stefan; Fischer, Andre; Liberski, Pawel P; Torres, Juan Maria; Ferrer, Isidre; Zerr, Inga

    2017-04-27

    Sporadic Creutzfeldt-Jakob disease (sCJD) is the most prevalent form of human prion disease and it is characterized by the presence of neuronal loss, spongiform degeneration, chronic inflammation and the accumulation of misfolded and pathogenic prion protein (PrP Sc ). The molecular mechanisms underlying these alterations are largely unknown, but the presence of intracellular neuronal calcium (Ca 2+ ) overload, a general feature in models of prion diseases, is suggested to play a key role in prion pathogenesis.Here we describe the presence of massive regulation of Ca 2+ responsive genes in sCJD brain tissue, accompanied by two Ca 2+ -dependent processes: endoplasmic reticulum stress and the activation of the cysteine proteases Calpains 1/2. Pathogenic Calpain proteins activation in sCJD is linked to the cleavage of their cellular substrates, impaired autophagy and lysosomal damage, which is partially reversed by Calpain inhibition in a cellular prion model. Additionally, Calpain 1 treatment enhances seeding activity of PrP Sc in a prion conversion assay. Neuronal lysosomal impairment caused by Calpain over activation leads to the release of the lysosomal protease Cathepsin S that in sCJD mainly localises in axons, although massive Cathepsin S overexpression is detected in microglial cells. Alterations in Ca 2+ homeostasis and activation of Calpain-Cathepsin axis already occur at pre-clinical stages of the disease as detected in a humanized sCJD mouse model.Altogether our work indicates that unbalanced Calpain-Cathepsin activation is a relevant contributor to the pathogenesis of sCJD at multiple molecular levels and a potential target for therapeutic intervention.

  10. TGF-ß Regulates Cathepsin Activation during Normal and Pathogenic Development.

    Science.gov (United States)

    Flanagan-Steet, Heather; Christian, Courtney; Lu, Po-Nien; Aarnio-Peterson, Megan; Sanman, Laura; Archer-Hartmann, Stephanie; Azadi, Parastoo; Bogyo, Matthew; Steet, Richard A

    2018-03-13

    Cysteine cathepsins play roles during development and disease beyond their function in lysosomal protein turnover. Here, we leverage a fluorescent activity-based probe (ABP), BMV109, to track cysteine cathepsins in normal and diseased zebrafish embryos. Using this probe in a model of mucolipidosis II, we show that loss of carbohydrate-dependent lysosomal sorting alters the activity of several cathepsin proteases. The data support a pathogenic mechanism where TGF-ß signals enhance the proteolytic processing of pro-Ctsk by modulating the expression of chondroitin 4-sulfate (C4-S). In MLII, elevated C4-S corresponds with TGF-ß-mediated increases in chst11 expression. Inhibiting chst11 impairs the proteolytic activation of Ctsk and alleviates the MLII phenotypes. These findings uncover a regulatory loop between TGF-ß signaling and Ctsk activation that is altered in the context of lysosomal disease. This work highlights the power of ABPs to identify mechanisms underlying pathogenic development in living animals. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

  11. The Diagnosis of Human Fascioliasis by Enzyme-Linked Immunosorbent Assay (ELISA) Using Recombinant Cathepsin L Protease

    Science.gov (United States)

    Gonzales Santana, Bibiana; Vasquez Camargo, Fabio; Parkinson, Michael

    2013-01-01

    Background Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Methodology/Principal findings Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. Conclusions/Significance A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this

  12. The diagnosis of human fascioliasis by enzyme-linked immunosorbent assay (ELISA) using recombinant cathepsin L protease.

    Science.gov (United States)

    Gonzales Santana, Bibiana; Dalton, John P; Vasquez Camargo, Fabio; Parkinson, Michael; Ndao, Momar

    2013-01-01

    Fascioliasis is a worldwide parasitic disease of domestic animals caused by helminths of the genus Fasciola. In many parts of the world, particularly in poor rural areas where animal disease is endemic, the parasite also infects humans. Adult parasites reside in the bile ducts of the host and therefore diagnosis of human fascioliasis is usually achieved by coprological examinations that search for parasite eggs that are carried into the intestine with the bile juices. However, these methods are insensitive due to the fact that eggs are released sporadically and may be missed in low-level infections, and fasciola eggs may be misclassified as other parasites, leading to problems with specificity. Furthermore, acute clinical symptoms as a result of parasites migrating to the bile ducts appear before the parasite matures and begins egg laying. A human immune response to Fasciola antigens occurs early in infection. Therefore, an immunological method such as ELISA may be a more reliable, easy and cheap means to diagnose human fascioliasis than coprological analysis. Using a panel of serum from Fasciola hepatica-infected patients and from uninfected controls we have optimized an enzyme-linked immunosorbent assay (ELISA) which employs a recombinant form of the major F. hepatica cathepsin L1 as the antigen for the diagnosis of human fascioliasis. We examined the ability of the ELISA test to discern fascioliasis from various other helminth and non-helminth parasitic diseases. A sensitive and specific fascioliasis ELISA test has been developed. This test is rapid and easy to use and can discriminate fasciola-infected individuals from patients harbouring other parasites with at least 99.9% sensitivity and 99.9% specificity. This test will be a useful standardized method not only for testing individual samples but also in mass screening programs to assess the extent of human fascioliasis in regions where this zoonosis is suspected.

  13. GILT expression in B cells diminishes cathepsin S steady-state protein expression and activity

    OpenAIRE

    Phipps-Yonas, Hannah; Semik, Vikki; Hastings, Karen Taraszka

    2012-01-01

    MHC class II-restricted Ag processing requires protein degradation in the endocytic pathway for the activation of CD4+ T cells. Gamma-interferon-inducible lysosomal thiol reductase (GILT) facilitates Ag processing by reducing protein disulfide bonds in this compartment. Lysosomal cysteine protease cathepsin S (CatS) contains disulfide bonds and mediates essential steps in MHC class II-restricted processing, including proteolysis of large polypeptides and cleavage of the invariant chain. We so...

  14. Vaccine potential of recombinant cathepsinL1G against Fasciola gigantica in mice.

    Science.gov (United States)

    Changklungmoa, Narin; Phoinok, Natthacha; Yencham, Chonthicha; Sobhon, Prasert; Kueakhai, Pornanan

    2016-08-15

    In this study, we characterized and investigated the vaccine potential of FgCatL1G against Fasciola gigantica infection in mice. Recombinant mature FgCatL1G (rmFgCatL1G) was expressed in Escherichia coli BL21. The vaccination was performed in Imprinting Control Region (ICR) mice (n=10) by subcutaneous injection with 50μg of rmFgCatL1G combined with Freund's adjuvant. Two weeks after the second boost, mice were infected with 15 metacercariae by the oral route. The percents of protection of rmFgCatL1G vaccine were estimated to be 56.5% and 58.3% when compared with non vaccinated-infected and adjuvant-infected controls, respectively. Antibodies in the immune sera of vaccinated mice were shown by immunoblot to react with the native FgCatL1s in the extract of all stages of parasites and rmFgCatL1H, recombinant pro - FgCatL1 (rpFgCatL1). By immunohistochemistry, the immune sera also reacted with FgCatL1s in the caecal epithelial cells of the parasites. The levels of IgG1 and IgG2a in the immune sera, which are indicative of Th2 and Th1 immune responses, were also increased with IgG1 predominating. The levels of serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) in rmFgCatL1G-immunized group showed no significant difference from the control groups, but pathological lesions of livers in rmFgCatL1G-immunized group showed significant decrease when compared to the control groups. This study indicates that rmFgCatL1G has a vaccine potential against F. gigantica in mice, and this potential will be tested in larger livestock animals. Copyright © 2016 Elsevier B.V. All rights reserved.

  15. Multiplex zymography captures stage-specific activity profiles of cathepsins K, L, and S in human breast, lung, and cervical cancer.

    Science.gov (United States)

    Chen, Binbin; Platt, Manu O

    2011-07-14

    Cathepsins K, L, and S are cysteine proteases upregulated in cancer and proteolyze extracellular matrix to facilitate metastasis, but difficulty distinguishing specific cathepsin activity in complex tissue extracts confounds scientific studies and employing them for use in clinical diagnoses. Here, we have developed multiplex cathepsin zymography to profile cathepsins K, L, and S activity in 10 μg human breast, lung, and cervical tumors by exploiting unique electrophoretic mobility and renaturation properties. Frozen breast, lung, and cervix cancer tissue lysates and normal organ tissue lysates from the same human patients were obtained (28 breast tissues, 23 lung tissues, and 23 cervix tissues), minced and homogenized prior to loading for cathepsin gelatin zymography to determine enzymatic activity. Cleared bands of cathepsin activity were identified and validated in tumor extracts and detected organ- and stage-specific differences in activity. Cathepsin K was unique compared to cathepsins L and S. It was significantly higher for all cancers even at the earliest stage tested (stage I for lung and cervix (n = 6, p zymography, yielded 100% sensitivity and specificity for 20 breast tissue samples tested (10 normal; 10 tumor) in part due to the consistent absence of cathepsin K in normal breast tissue across all patients. To summarize, this sensitive assay provides quantitative outputs of cathepsins K, L, and S activities from mere micrograms of tissue and has potential use as a supplement to histological methods of clinical diagnoses of biopsied human tissue.

  16. Improving serodiagnosis of human and canine leishmaniasis with recombinant Leishmania braziliensis cathepsin l-like protein and a synthetic peptide containing its linear B-cell epitope.

    Directory of Open Access Journals (Sweden)

    Daniel Menezes-Souza

    2015-01-01

    Full Text Available The early and correct diagnosis of human leishmaniasis is essential for disease treatment. Another important step in the control of visceral leishmaniasis is the identification of infected dogs, which are the main domestic reservoir of L. infantum. Recombinant proteins and synthetic peptides based on Leishmania genes have emerged as valuable targets for serodiagnosis due to their increased sensitivity, specificity and potential for standardization. Cathepsin L-like genes are surface antigens that are secreted by amastigotes and have little similarity to host proteins, factors that enable this protein as a good target for serodiagnosis of the leishmaniasis.We mapped a linear B-cell epitope within the Cathepsin L-like protein from L. braziliensis. A synthetic peptide containing the epitope and the recombinant protein was evaluated for serodiagnosis of human tegumentary and visceral leishmaniasis, as well as canine visceral leishmaniasis.The recombinant protein performed best for human tegumentary and canine visceral leishmaniasis, with 96.30% and 89.33% accuracy, respectively. The synthetic peptide was the best to discriminate human visceral leishmaniasis, with 97.14% specificity, 94.55% sensitivity and 96.00% accuracy. Comparison with T. cruzi-infected humans and dogs suggests that the identified epitope is specific to Leishmania parasites, which minimizes the likelihood of cross-reactions.

  17. Manipulating substrate and pH in zymography protocols selectively distinguishes cathepsins K, L, S, and V activity in cells and tissues.

    Science.gov (United States)

    Wilder, Catera L; Park, Keon-Young; Keegan, Philip M; Platt, Manu O

    2011-12-01

    Cathepsins K, L, S, and V are cysteine proteases that have been implicated in tissue-destructive diseases such as atherosclerosis, tumor metastasis, and osteoporosis. Among these four cathepsins are the most powerful human collagenases and elastases, and they share 60% sequence homology. Proper quantification of mature, active cathepsins has been confounded by inhibitor and reporter substrate cross-reactivity, but is necessary to develop properly dosed therapeutic applications. Here, we detail a method of multiplex cathepsin zymography to detect and distinguish the activity of mature cathepsins K, L, S, and V by exploiting differences in individual cathepsin substrate preferences, pH effects, and electrophoretic mobility under non-reducing conditions. Specific identification of cathepsins K, L, S, and V in one cell/tissue extract was obtained with cathepsin K (37 kDa), V (35 kDa), S (25 kDa), and L (20 kDa) under non-reducing conditions. Cathepsin K activity disappeared and V remained when incubated at pH 4 instead of 6. Application of this antibody free, species independent, and medium-throughput method was demonstrated with primary human monocyte-derived macrophages and osteoclasts, endothelial cells stimulated with inflammatory cytokines, and normal and cancer lung tissues, which identified elevated cathepsin V in lung cancer. Copyright © 2011 Elsevier Inc. All rights reserved.

  18. Effects of Chilling and Partial Freezing on Rigor Mortis Changes of Bighead Carp (Aristichthys nobilis) Fillets: Cathepsin Activity, Protein Degradation and Microstructure of Myofibrils.

    Science.gov (United States)

    Lu, Han; Liu, Xiaochang; Zhang, Yuemei; Wang, Hang; Luo, Yongkang

    2015-12-01

    To investigate the effects of chilling and partial freezing on rigor mortis changes in bighead carp (Aristichthys nobilis), pH, cathepsin B, cathepsin B+L activities, SDS-PAGE of sarcoplasmic and myofibrillar proteins, texture, and changes in microstructure of fillets at 4 °C and -3 °C were determined at 0, 2, 4, 8, 12, 24, 48, and 72 h after slaughter. The results indicated that pH of fillets (6.50 to 6.80) was appropriate for cathepsin function during the rigor mortis. For fillets that were chilled and partially frozen, the cathepsin activity in lysosome increased consistently during the first 12 h, followed by a decrease from the 12 to 24 h, which paralleled an increase in activity in heavy mitochondria, myofibrils and sarcoplasm. There was no significant difference in cathepsin activity in lysosomes between fillets at 4 °C and -3 °C (P > 0.05). Partially frozen fillets had greater cathepsin activity in heavy mitochondria than chilled samples from the 48 to 72 h. In addition, partially frozen fillets showed higher cathepsin activity in sarcoplasm and lower cathepsin activity in myofibrils compared with chilled fillets. Correspondingly, we observed degradation of α-actinin (105 kDa) by cathepsin L in chilled fillets and degradation of creatine kinase (41 kDa) by cathepsin B in partially frozen fillets during the rigor mortis. The decline of hardness for both fillets might be attributed to the accumulation of cathepsin in myofibrils from the 8 to 24 h. The lower cathepsin activity in myofibrils for fillets that were partially frozen might induce a more intact cytoskeletal structure than fillets that were chilled. © 2015 Institute of Food Technologists®

  19. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae): A Putative Target for Control of Citrus Huanglongbing.

    Science.gov (United States)

    Ferrara, Taíse Fernanda da Silva; Schneider, Vanessa Karine; Kishi, Luciano Takeshi; Carmona, Adriana Karaoglanovic; Alves, Marcio Fernando Madureira; Belasque-Júnior, Jose; Rosa, José César; Hunter, Wayne Brian; Henrique-Silva, Flávio; Soares-Costa, Andrea

    2015-01-01

    Huanglonbing (HLB) is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas) and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB). DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM) and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM). The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM) and CaneCPI-4 (Ki = 0.05 nM) and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM). RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

  20. Characterization of a Recombinant Cathepsin B-Like Cysteine Peptidase from Diaphorina citri Kuwayama (Hemiptera: Liviidae: A Putative Target for Control of Citrus Huanglongbing.

    Directory of Open Access Journals (Sweden)

    Taíse Fernanda da Silva Ferrara

    Full Text Available Huanglonbing (HLB is one of the most destructive disease affecting citrus plants. The causal agent is associated with the phloem-limited bacterium Candidatus Liberibacter asiaticus (CLas and the psyllid Diaphorina citri, vector of disease, that transmits the bacterium associated with HLB. The control of disease can be achieved by suppressing either the bacterium or the vector. Among the control strategies for HLB disease, one of the widely used consists in controlling the enzymes of the disease vector, Diaphorina citri. The insect Diaphorina citri belongs to the order Hemiptera, which frequently have cysteine peptidases in the gut. The importance of this class of enzymes led us to search for enzymes in the D. citri transcriptome for the establishment of alternatives strategies for HLB control. In this study, we reported the identification and characterization of a cathepsin B-like cysteine peptidase from D. citri (DCcathB. DCcathB was recombinantly expressed in Pichia pastoris, presenting a molecular mass of approximately 50 kDa. The enzyme hydrolyzed the fluorogenic substrate Z-F-R-AMC (Km = 23.5 μM and the selective substrate for cathepsin B, Z-R-R-AMC (Km = 6.13 μM. The recombinant enzyme was inhibited by the cysteine protease inhibitors E64 (IC50 = 0.014 μM and CaneCPI-4 (Ki = 0.05 nM and by the selective cathepsin B inhibitor CA-074 (IC50 = 0.095 nM. RT-qPCR analysis revealed that the expression of the DCcathB in nymph and adult was approximately 9-fold greater than in egg. Moreover, the expression of this enzyme in the gut was 175-fold and 3333-fold higher than in the remaining tissues and in the head, respectively, suggesting that DCcathB can be a target for HLB control.

  1. Cathepsin activities and thermal properties of Nile tilapia (Oreochromis niloticus meat during ambient storage

    Directory of Open Access Journals (Sweden)

    Tulakhun Nonthaput

    2017-06-01

    Full Text Available Understanding the postmortem changes at ambient aquatic temperature can be useful for estimating the time of death in environmental forensic studies when little information is available. Muscle degradation was investigated in Nile tilapia (Oreochromis niloticus in terms of the specific activities of cathepsins (B, H and L and the scavenging activities and thermal transition properties of myosin and actin, to assess postmortem changes with time (0, 1, 2, 4, 8, 12, 24 and 48 h after death. The study results are relevant to ambient temperatures in Thailand, (about 30 °C. The specific activities of the three cathepsin enzymes increased significantly with postmortem time (p < 0.05 and had a highly significant positive relationship (r = 0.987−0.997, p < 0.01, n = 32. Cathepsin H had the lowest specific activity and exhibited a different type of time profile. Its lowest specific activity was observed at 8 h, which indicated a significant role at this point in time after death. The radical scavenging activities substantially decreased with the time since death, especially within the first 1 h, while no changes occurred from 2 to 8 h, or from 12 to 24 h. The thermal properties of myosin and actin were observed up to a 24 h delay. The degradation of each protein fluctuated with the delay time; actin was more sensitive to postmortem delay than myosin. Overall, the findings from the current study might be used as primary data to estimate the time of death of an aquatic animal. A potential application is for environmental forensics in relation to fish kill events associated with pollution crimes or the mass death of exported fish under transportation insurance, as well as in animal cruelty investigations.

  2. In Vivo Molecular Imaging of Cathepsin and Matrix Metalloproteinase Activity Discriminates between Arthritic and Osteoarthritic Processes in Mice

    Directory of Open Access Journals (Sweden)

    Eline A. Vermeij

    2014-01-01

    Full Text Available Rheumatoid arthritis (RA and osteoarthritis (OA are serologically and clinically distinctive, but at the local level, both diseases have many molecular pathways in common. In vivo molecular imaging can unravel the local pathologic processes involved in both diseases. In this study, we investigated matrix metalloproteinase (MMP and cathepsin activity during cartilage destruction, in an RA and an OA mouse model, using biophotonic imaging of substrate-based probes. Mice with collagen-induced arthritis (CIA or destabilization of the medial meniscus (DMM were imaged using near-infrared fluorescent probes, activated by several cathepsins or MMPs. Fluorescence signal intensity was compared to synovial gene expression, histology, and cartilage staining of a neoepitope of aggrecan cleaved by MMPs with the amino acids DIPEN. Increased cathepsin and MMP activity was seen during CIA, whereas the DMM model only showed increased MMP activity. DIPEN expression was seen only during CIA. A possible explanation can be differences in gene expressions; MMP3 and -13, known to produce DIPEN neoepitopes, were upregulated in the CIA model, whereas MMP12, known to be involved in elastin degradation and chemokine inhibition, was upregulated in the DMM model. Thus, molecular imaging showed no cathepsin activity at the time of cartilage damage in the DMM model, whereas both cathepsins and MMPs are active in the CIA model during disease progression.

  3. A novel approach for reliable detection of cathepsin S activities in mouse antigen presenting cells.

    Science.gov (United States)

    Steimle, Alex; Kalbacher, Hubert; Maurer, Andreas; Beifuss, Brigitte; Bender, Annika; Schäfer, Andrea; Müller, Ricarda; Autenrieth, Ingo B; Frick, Julia-Stefanie

    2016-05-01

    Cathepsin S (CTSS) is a eukaryotic protease mostly expressed in professional antigen presenting cells (APCs). Since CTSS activity regulation plays a role in the pathogenesis of various autoimmune diseases like multiple sclerosis, atherosclerosis, Sjögren's syndrome and psoriasis as well as in cancer progression, there is an ongoing interest in the reliable detection of cathepsin S activity. Various applications have been invented for specific detection of this enzyme. However, most of them have only been shown to be suitable for human samples, do not deliver quantitative results or the experimental procedure requires technical equipment that is not commonly available in a standard laboratory. We have tested a fluorogen substrate, Mca-GRWPPMGLPWE-Lys(Dnp)-DArg-NH2, that has been described to specifically detect CTSS activities in human APCs for its potential use for mouse samples. We have modified the protocol and thereby offer a cheap, easy, reproducible and quick activity assay to detect CTSS activities in mouse APCs. Since most of basic research on CTSS is performed in mice, this method closes a gap and offers a possibility for reliable and quantitative CTSS activity detection that can be performed in almost every laboratory. Copyright © 2016. Published by Elsevier B.V.

  4. Cathepsin B inhibitory activities of three new phthalate derivatives isolated from seahorse, Hippocampus Kuda Bleeler.

    Science.gov (United States)

    Li, Yong; Qian, Zhong-Ji; Kim, Se-Kwon

    2008-12-01

    Three new phthalate acid derivatives, 2,12-diethyl-11-methylhexadecyl 2-ethyl-11-methylhexadecyl phthalate (1), 2-ethyldecyl 2-ethylundecyl phthalate (2), and bis(2-ethyldodecyl) phthalate (3), were isolated from seahorse, Hippocampus Kuda Bleeler, together with a known natural analog bis(2-ethylheptyl) phthalate (4). The structures of these compounds were elucidated mainly by means of the comprehensive analysis of their NMR spectroscopic data. The four phthalate derivatives showed dose-dependent cathepsin B inhibitions activities with IC(50) values of 0.13 mM (1), 0.21 mM (2), 0.18 mM (3), and 0.29 mM (4), respectively.

  5. Recombinant snake venom prothrombin activators

    OpenAIRE

    L?vgren, Ann

    2012-01-01

    Three prothrombin activators; ecarin, which was originally isolated from the venom of the saw-scaled viper Echis carinatus, trocarin from the rough-scaled snake Tropidechis carinatus, and oscutarin from the Taipan snake Oxyuranus scutellatus, were expressed in mammalian cells with the purpose to obtain recombinant prothrombin activators that could be used to convert prothrombin to thrombin. We have previously reported that recombinant ecarin can efficiently generate thrombin without the need ...

  6. Impact of the Enhanced Permeability and Retention (EPR Effect and Cathepsins Levels on the Activity of Polymer-Drug Conjugates

    Directory of Open Access Journals (Sweden)

    Amit K. Rajora

    2014-08-01

    Full Text Available Polymer-drug conjugates have demonstrated clinical potential in the context of anticancer therapy. However, such promising results have, to date, failed to translate into a marketed product. Polymer-drug conjugates rely on two factors for activity: (i the presence of a defective vasculature, for passive accumulation of this technology into the tumour tissue (enhanced permeability and retention (EPR effect and (ii the presence of a specific trigger at the tumour site, for selective drug release (e.g., the enzyme cathepsin B. Here, we retrospectively analyse literature data to investigate which tumour types have proved more responsive to polymer-drug conjugates and to determine correlations between the magnitude of the EPR effect and/or expression of cathepsin B. Lung, breast and ovarian cancers showed the highest response rate (30%, 47% and 41%, respectively for cathepsin-activated conjugates and 31%, 43%, 40%, across all conjugates. An analysis of literature data on cathepsin content in various tumour types showed that these tumour types had high cathepsin content (up to 3835 ng/mg for lung cancer, although marked heterogeneity was observed across different studies. In addition, these tumour types were also reported as having a high EPR effect. Our results suggest that a pre-screening of patient population could bring a more marked clinical benefit.

  7. Design of a highly selective quenched activity-based probe and its application in dual color imaging studies of cathepsin S activity localization.

    Science.gov (United States)

    Oresic Bender, Kristina; Ofori, Leslie; van der Linden, Wouter A; Mock, Elliot D; Datta, Gopal K; Chowdhury, Somenath; Li, Hao; Segal, Ehud; Sanchez Lopez, Mateo; Ellman, Jonathan A; Figdor, Carl G; Bogyo, Matthew; Verdoes, Martijn

    2015-04-15

    The cysteine cathepsins are a group of 11 proteases whose function was originally believed to be the degradation of endocytosed material with a high degree of redundancy. However, it has become clear that these enzymes are also important regulators of both health and disease. Thus, selective tools that can discriminate between members of this highly related class of enzymes will be critical to further delineate the unique biological functions of individual cathepsins. Here we present the design and synthesis of a near-infrared quenched activity-based probe (qABP) that selectively targets cathepsin S which is highly expressed in immune cells. Importantly, this high degree of selectivity is retained both in vitro and in vivo. In combination with a new green-fluorescent pan-reactive cysteine cathepsin qABP we performed dual color labeling studies in bone marrow-derived immune cells and identified vesicles containing exclusively cathepsin S activity. This observation demonstrates the value of our complementary cathepsin probes and provides evidence for the existence of specific localization of cathepsin S activity in dendritic cells.

  8. Unnatural amino acids increase activity and specificity of synthetic substrates for human and malarial cathepsin C.

    Science.gov (United States)

    Poreba, Marcin; Mihelic, Marko; Krai, Priscilla; Rajkovic, Jelena; Krezel, Artur; Pawelczak, Malgorzata; Klemba, Michael; Turk, Dusan; Turk, Boris; Latajka, Rafal; Drag, Marcin

    2014-04-01

    Mammalian cathepsin C is primarily responsible for the removal of N-terminal dipeptides and activation of several serine proteases in inflammatory or immune cells, while its malarial parasite ortholog dipeptidyl aminopeptidase 1 plays a crucial role in catabolizing the hemoglobin of its host erythrocyte. In this report, we describe the systematic substrate specificity analysis of three cathepsin C orthologs from Homo sapiens (human), Bos taurus (bovine) and Plasmodium falciparum (malaria parasite). Here, we present a new approach with a tailored fluorogenic substrate library designed and synthesized to probe the S1 and S2 pocket preferences of these enzymes with both natural and a broad range of unnatural amino acids. Our approach identified very efficiently hydrolyzed substrates containing unnatural amino acids, which resulted in the design of significantly better substrates than those previously known. Additionally, in this study significant differences in terms of the structures of optimal substrates for human and malarial orthologs are important from the therapeutic point of view. These data can be also used for the design of specific inhibitors or activity-based probes.

  9. Analysis of cathepsin and furin proteolytic enzymes involved in viral fusion protein activation in cells of the bat reservoir host.

    Directory of Open Access Journals (Sweden)

    Farah El Najjar

    Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.

  10. Lipotoxicity Mediated Cell Dysfunction and Death Involves Lysosomal Membrane Permeabilization and Cathepsin L Activity

    Science.gov (United States)

    Almaguel, Frankis G.; Liu, Jo-Wen; Pacheco, Fabio J.; De Leon, Daisy; Casiano, Carlos A.; De Leon, Marino

    2010-01-01

    Lipotoxicity, which is triggered when cells are exposed to elevated levels of free fatty acids, involves cell dysfunction and apoptosis and is emerging as an underlying factor contributing to various pathological conditions including disorders of the central nervous system and diabetes. We have shown that palmitic acid (PA)-induced lipotoxicity (PA-LTx) in nerve growth factor-differentiated PC12 (NGFDPC12) cells is linked to an augmented state of cellular oxidative stress (ASCOS) and apoptosis, and that these events are inhibited by docosahexanoic acid (DHA). The mechanisms of PA-LTx in nerve cells are not well understood, but our previous findings indicate that it involves ROS generation, mitochondrial membrane permeabilization (MMP), and caspase activation. The present study used nerve growth factor differentiated PC12 cells (NGFDPC12 cells) and found that lysosomal membrane permeabilization (LMP) is an early event during PA-induced lipotoxicity that precedes MMP and apoptosis. Cathepsin L, but not cathepsin B, is an important contributor in this process since its pharmacological inhibition significantly attenuated LMP, MMP, and apoptosis. In addition, co-treatment of NGFDPC12 cells undergoing lipotoxicity with DHA significantly reduced LMP, suggesting that DHA acts by antagonizing upstream signals leading to lysosomal dysfunction. These results suggest that LMP is a key early mediator of lipotoxicity, and underscore the value of interventions targeting upstream signals leading to LMP for the treatment of pathological conditions associated with lipotoxicity. PMID:20043885

  11. Cathepsin H indirectly regulates morphogenetic protein-4 (BMP-4) in various human cell lines

    Science.gov (United States)

    Rojnik, Matija; Jevnikar, Zala; Mirkovic, Bojana; Janes, Damjan; Zidar, Nace; Kikelj, Danijel; Kos, Janko

    2011-01-01

    Background Cathepsin H is a cysteine protease considered to play a major role in tumor progression, however, its precise function in tumorigenesis is unclear. Cathepsin H was recently proposed to be involved in processing of bone morphogenetic protein 4 (BMP-4) in mice. In order to clarify whether cathepsin H also regulates BMP-4 in humans, its impact on BMP-4 expression, processing and degradation was investigated in prostate cancer (PC-3), osteosarcoma (HOS) and pro-monocytic (U937) human cell lines. Materials and methods BMP-4 expression was founded to be regulated by cathepsin H using PCR array technology and confirmed by real time PCR. Immunoassays including Western blot and confocal microscopy were used to evaluate the influence of cathepsin H on BMP-4 processing. Results In contrast to HOS, the expression of BMP-4 mRNA in U937 and PC3 cells was significantly decreased by cathepsin H. The different regulation of BMP-4 synthesis could be associated with the absence of the mature 28 kDa cathepsin H form in HOS cells, where only the intermediate 30 kDa form was observed. No co-localization of BMP-4 and cathepsin H was observed in human cell lines and the multistep processing of BMP-4 was not altered in the presence of specific cathepsin H inhibitor. Isolated cathepsin H does not cleave mature recombinant BMP-4, neither with its amino- nor its endopeptidase activity. Conclusions Our results exclude direct proteolytic processing of BMP-4 by cathepsin H, however, they provide support for its involvement in the regulation of BMP-4 expression. PMID:22933963

  12. Single- and Double-Headed Chemical Probes for Detection of Active Cathepsin D in a Cancer Cell Proteome

    Czech Academy of Sciences Publication Activity Database

    Nussbaumerová, Martina; Srp, Jaroslav; Máša, Martin; Hradilek, Martin; Šanda, Miloslav; Reiniš, Milan; Horn, Martin; Mareš, Michael

    2010-01-01

    Roč. 11, č. 11 (2010), s. 1538-1541 ISSN 1439-4227 R&D Projects: GA AV ČR IAA400550705 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : cathepsin D * cancer * activity-based probes Subject RIV: CE - Biochemistry Impact factor: 3.945, year: 2010

  13. Cathepsin activities and membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling.

    Science.gov (United States)

    Zhang, T; Rawson, D M; Tosti, L; Carnevali, O

    2008-04-01

    This study investigated enzymatic activity of cathepsins and the membrane integrity of zebrafish (Danio rerio) oocytes after freezing to -196 degrees C using controlled slow cooling. Stage III oocytes (>0.5mm), obtained through dissection of anaesthetised female fish and desegregation of ovarian cumulus, were exposed to 2M methanol or 2M DMSO (both prepared in Hank's medium) for 30min at 22 degrees C before being loaded into 0.5ml plastic straws and placed into a programmable cooler. After controlled slow freezing, samples were plunged into liquid nitrogen (LN) and held for at least 10min, and thawed by immersing straws into a 27 degrees C water bath for 10s. Thawed oocytes were washed twice in Hank's medium. Cathepsin activity and membrane integrity of oocytes were assessed both after cryoprotectant treatment at 22 degrees C and after freezing in LN. Cathepsin B and L colorimetric analyses were performed using substrates Z-Arg-ArgNNap and Z-Phe-Arg-4MbetaNA-HCl, respectively, and 2-naphthylamine and 4-methoxy-2-naphthylamine were used as standards. Cathepsin D activity was performed by analysing the level of hydrolytic action on haemoglobin. Oocytes membrane integrity was assessed using 0.2% Trypan blue staining for 5min. Analysis of cathepsin activities showed that whilst the activity of cathepsin B and D was not affected by 2M DMSO treatment, their activity was lowered when treated with 2M methanol. Following freezing to -196 degrees C, the activity of all cathepsins (B, D and L) was significantly decreased in both 2M DMSO and 2M methanol. Trypan blue staining showed that 63.0+/-11.3% and 72.7+/-5.2% oocytes membrane stayed intact after DMSO and methanol treatment for 30min at 22 degrees C, respectively, whilst 14.9+/-2.6% and 1.4+/-0.8% stayed intact after freezing in DMSO and methanol to -196 degrees C. The results indicate that cryoprotectant treatment and freezing modified the activities of lysosomal enzymes involved in oocyte maturation and yolk

  14. Excessive activity of cathepsin K is associated with cartilage defects in a zebrafish model of mucolipidosis II

    Directory of Open Access Journals (Sweden)

    Aaron C. Petrey

    2012-03-01

    The severe pediatric disorder mucolipidosis II (ML-II; also known as I-cell disease is caused by defects in mannose 6-phosphate (Man-6-P biosynthesis. Patients with ML-II exhibit multiple developmental defects, including skeletal, craniofacial and joint abnormalities. To date, the molecular mechanisms that underlie these clinical manifestations are poorly understood. Taking advantage of a zebrafish model of ML-II, we previously showed that the cartilage morphogenesis defects in this model are associated with altered chondrocyte differentiation and excessive deposition of type II collagen, indicating that aspects of development that rely on proper extracellular matrix homeostasis are sensitive to decreases in Man-6-P biosynthesis. To further investigate the molecular bases for the cartilage phenotypes, we analyzed the transcript abundance of several genes in chondrocyte-enriched cell populations isolated from wild-type and ML-II zebrafish embryos. Increased levels of cathepsin and matrix metalloproteinase (MMP transcripts were noted in ML-II cell populations. This increase in transcript abundance corresponded with elevated and sustained activity of several cathepsins (K, L and S and MMP-13 during early development. Unlike MMP-13, for which higher levels of protein were detected, the sustained activity of cathepsin K at later stages seemed to result from its abnormal processing and activation. Inhibition of cathepsin K activity by pharmacological or genetic means not only reduced the activity of this enzyme but led to a broad reduction in additional protease activity, significant correction of the cartilage morphogenesis phenotype and reduced type II collagen staining in ML-II embryos. Our findings suggest a central role for excessive cathepsin K activity in the developmental aspects of ML-II cartilage pathogenesis and highlight the utility of the zebrafish system to address the biochemical underpinnings of metabolic disease.

  15. Cathepsin D inhibitors

    Directory of Open Access Journals (Sweden)

    M. Gacko

    2007-11-01

    Full Text Available Inhibitors of cathepsin D belong to chemical compounds that estrify carboxyl groups of the Asp33 and Asp231residues of its catalytic site, penta-peptides containing statin, i.e. the amino acid similar in structure to the tetraedric indirectproduct, and polypeptides found in the spare organs of many plants and forming permanent noncovalent complexes withcathepsin. Cathepsin D activity is also inhibited by alpha2-macroglobulin and antibodies directed against this enzyme.Methods used to determine the activity and concentration of these inhibitors and their analytical, preparative and therapeuticapplications are discussed.

  16. Recombinant Cyclophilins Lack Nuclease Activity

    OpenAIRE

    Manteca, Angel; Sanchez, Jesus

    2004-01-01

    Several single-domain prokaryotic and eukaryotic cyclophilins have been identified as also being unspecific nucleases with a role in DNA degradation during the lytic processes that accompany bacterial cell death and eukaryotic apoptosis. Evidence is provided here that the supposed nuclease activity of human and bacterial recombinant cyclophilins is due to contamination of the proteins by the host Escherichia coli endonuclease and is not an intrinsic property of these proteins.

  17. Affinity purification of recombinant human plasminogen activator ...

    African Journals Online (AJOL)

    Affinity purification of recombinant human plasminogen activator from ... Screening antibody was performed using rhPA milk in an ELISA-elution assay. ... useful for purifying other tPA mutants or other novel recombinant milkderived proteins.

  18. Evaluation of matrix metalloproteinase and cysteine cathepsin activity in dentin hybrid layer by gelatin zymography.

    Science.gov (United States)

    Mahalaxmi, Sekar; Madhubala, Manavalan Madhana; Jayaraman, Mahendran; Sathyakumar, Shanmugasundaram

    2016-01-01

    The aim of this study was to comparatively assess the gelatinolytic activity of matrix metalloproteinases(MMPs) and Cysteine Cathepsins (CCs) in the adhesive interface using etch and rinse adhesive at different time intervals using zymographic technique. Twenty freshly extracted non-carious human third molars were used in this study. Occlusal surfaces were ground flat and 1mm thick horizontal dentin slabs were obtained from each tooth using a diamond disc. The dentin surface was polished with 600-grit silicon-carbide paper. Five out of 20 samples were directly pulverized. In the remaining fifteen samples, the dentin was etched and adhesive was applied and light cured according to the manufacturer's instructions. A 1mm thick flowable composite was build up and light cured. Bonded specimens were cut vertically into 3 to 4 dentin slabs by means of diamond disc to expose the adhesive/dentin interfaces. These were then ground down to 500 µm thick resin-dentin interface using a hard tissue microtome. These sections were then pulverised into powder. Following this, every five samples were subjected to zymographic analysis after 1 day, 7 days and 21 days. Zymograms showed clear, thicker bands on all three isoforms in the etched samples compared to control samples at 1st and 7th day intervals and became inactive at 21st day for all three isoforms. MMP 9 activity was relatively higher when compared to CCs and MMP 2. Etch and rinse adhesive activated MMPs and CCs within the hybrid layer that remained active till 7th day and no gelatinolytic activity was found on 21st day and MMPs are more active compared to CCs and MMP-2.

  19. Endothelium-dependent relaxation induced by cathepsin G in porcine pulmonary arteries

    Science.gov (United States)

    Glusa, Erika; Adam, Christine

    2001-01-01

    Serine proteinases elicit profound cellular effects in various tissues mediated by activation of proteinase-activated receptors (PAR). In the present study, we investigated the vascular effects of cathepsin G, a serine proteinase that is present in the azurophil granules of leukocytes and is known to activate several cells that express PARs. In prostaglandin F2α (3 μM)-precontracted rings from porcine pulmonary arteries with intact endothelium, cathepsin G caused concentration-dependent relaxant responses (pEC50=9.64±0.12). The endothelium-dependent relaxant effect of cathepsin G could also be demonstrated in porcine coronary arteries (pEC50=9.23±0.07). In pulmonary arteries the cathepsin G-induced relaxation was inhibited after blockade of nitric oxide synthesis by L-NAME (200 μM) and was absent in endothelium-denuded vessels. Bradykinin- and cathepsin G-induced relaxant effects were associated with a 5.7 fold and 2.4 fold increase in the concentration of cyclic GMP, respectively. Compared with thrombin and trypsin, which also produced an endothelium-dependent relaxation in pulmonary arteries, cathepsin G was 2.5 and four times more potent, respectively. Cathepsin G caused only small homologous desensitization. In cathepsin G-challenged vessels, thrombin was still able to elicit a relaxant effect. The effects of cathepsin G were blocked by soybean trypsin inhibitor (IC50=0.043 μg ml−1), suggesting that proteolytic activity is essential for induction of relaxation. Recombinant acetyl-eglin C proved to be a potent inhibitor (IC50=0.14 μg ml−1) of the cathepsin G effect, whereas neither indomethacin (3 μM) nor the thrombin inhibitor hirudin (5 ATU ml−1) elicited any inhibitory activity. Due to their polyanionic structure defibrotide (IC50=0.11 μg ml−1), heparin (IC50=0.48 μg ml−1) and suramin (IC50=1.85 μg ml−1) diminished significantly the relaxation in response to the basic protein cathepsin G. In conclusion, like

  20. Activation route of the Schistosoma mansoni cathepsin B1 drug target

    Czech Academy of Sciences Publication Activity Database

    Jílková, Adéla; Horn, Martin; Řezáčová, Pavlína; Marešová, Lucie; Fajtová, Pavla; Brynda, Jiří; Vondrášek, Jiří; McKerrow, J. H.; Caffrey, C. R.; Mareš, Michael

    2015-01-01

    Roč. 22, č. 1 (2015), s. 39 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : Schistosoma mansoni * cathepsin B1 * sulfated polysaccharides Subject RIV: CE - Biochemistry

  1. Downregulation of cathepsin G reduces the activation of CD4+ T cells in murine autoimmune diabetes.

    Science.gov (United States)

    Zou, Fang; Lai, Xiaoyang; Li, Jing; Lei, Shuihong; Hu, Lei

    2017-01-01

    Type 1 diabetes mellitus (T1DM) is an autoimmune disease due to progressive injury of islet cells mediated by T lymphocytes (T cells). Our previous studies have shown that only cathepsin G (CatG), not other proteases, is involved in the antigen presentation of proinsulin, and if the presentation is inhibited, the activation of CD4+ T cells induced by proinsulin is alleviated in T1DM patients, and CatG-specific inhibitor reduces the activation of CD4+ cells induced by proinsulin in T1DM patients. Therefore, we hypothesize that CatG may play an important role in the activation of CD4+ T cells in T1DM. To this end, mouse studies were conducted to demonstrate that CatG impacts the activation of CD4+ T cells in non-obese diabetic (NOD) mice. CatG gene expression and the activation of CD4+ T cells were examined in NOD mice. The effect of CatG inhibitor was investigated in NOD mice on the activation of CD4+ T cells, islet β cell function, islet inflammation and β-cell apoptosis. Furthermore, NOD mice were injected with CatG siRNA in early stage to observe the effect of CatG knockdown on the activation status of CD4+ T cells and the progression of diabetes. During the pathogenesis of diabetes, the expression level of CatG in NOD mice gradually increased and the CD4+ T cells were gradually activated, resulting in more TH1 cells and less TH2 and Treg cells. Treatment with CatG-specific inhibitor reduced the blood glucose level, improved the function of islet β cells and reduced the activation of CD4+ T cells. Early application of CatG siRNA improved the function of islet β cells, reduced islet inflammation and β cell apoptosis, and lowered the activation level of CD4+ T cells, thus slowing down the progression of diabetes.

  2. New method to discriminate between cathepsin B and cathepsin L in crude extracts from fish muscle based on a simple acidification procedure

    DEFF Research Database (Denmark)

    Godiksen, Helene; Nielsen, Henrik Hauch

    2007-01-01

    A new and simple method to distinguish between cathepsin B and cathepsin L in crude extracts of herring (Clupea harengus) muscle has been established. An acid treatment of crude extracts (exposed to pH 3 for 5 min) activated a latent form of cathepsin L and inactivated cathepsin B. Furthermore......, in neutral crude extract, the hydrolysis of benzyloxycarbonyl-L-phenylalanyl-L-arginyl-4-methylcoumarine (Z-Phe-Arg-MCA) (cathepsin B and cathepsin L substrates) was between 0% and 15% of the hydrolysis of benzyloxycarbonyl-L-arginyl-L-arginyl-7-amino-4-methylcoumarine (Z-Arg-Arg-MCA; cathepsin B substrate......). Cathepsin B activity is measured in neutral extract using the specific cathepsin B substrate Z-Arg-Arg-MCA and cathepsin L activity is measured in acid-treated extract with Z-Phe-Arg-MCA as substrate. The specific cathepsin B inhibitor, CA-074, did not inhibit the Z-Arg-Arg-MCA significantly without...

  3. Exploration of peptides that fit into the thermally vibrating active site of cathepsin K protease by alternating artificial intelligence and molecular simulation

    Science.gov (United States)

    Nishiyama, Katsuhiko

    2017-08-01

    Eighteen tripeptides that fit into the thermally vibrating active site of cathepsin K were discovered by alternating artificial intelligence and molecular simulation. The 18 tripeptides fit the active site better than the cysteine protease inhibitor E64, and a better inhibitor of cathepsin K could be designed considering these tripeptides. Among the 18 tripeptides, Phe-Arg-Asp and Tyr-Arg-Asp fit the active site the best and their structural similarity should be considered in the design process. Interesting factors emerged from the structure of the decision tree, and its structural information will guide exploration of potential inhibitor molecules for proteases.

  4. ErbB2-Driven Breast Cancer Cell Invasion Depends on a Complex Signaling Network Activating Myeloid Zinc Finger-1-Dependent Cathepsin B Expression

    DEFF Research Database (Denmark)

    Rafn, Bo; Nielsen, Christian Thomas Friberg; Andersen, Sofie Hagel

    2012-01-01

    Aberrant ErbB2 receptor tyrosine kinase activation in breast cancer is strongly linked to an invasive disease. The molecular basis of ErbB2-driven invasion is largely unknown. We show that cysteine cathepsins B and L are elevated in ErbB2 positive primary human breast cancer and function...... as effectors of ErbB2-induced invasion in vitro. We identify Cdc42-binding protein kinase beta, extracellular regulated kinase 2, p21-activated protein kinase 4, and protein kinase C alpha as essential mediators of ErbB2-induced cysteine cathepsin expression and breast cancer cell invasiveness. The identified...

  5. Molecular requirements for radiation-activated recombination

    International Nuclear Information System (INIS)

    Stevens, Craig W.; Zeng Ming; Stamato, Thomas; Cerniglia, George

    1997-01-01

    Purpose/Objective: The major stumbling block to successful gene therapy today is poor gene transfer. We hypothesized that ionizing radiation might activate cellular recombination, and so improve stable gene transfer. We further hypothesized that known DNA-damage-repair proteins might also be important in radiation-activated recombination. Materials and Methods: The effect of irradiation on stable gene transfer efficiency was determined in human (A549 and 39F) and rodent (NIH/3T3) cell lines. Continuous low dose rate and multiple radiation fractions were also tested. Nuclear extracts were made and the effect of irradiation on inter-plasmid recombination/ligation determined. Multiple DNA damage-repair deficient cell lines were tested for radiation-activated recombination. Results: A significant radiation dose-dependent improvement in stable plasmid transfection (by as much as 1300 fold) is demonstrated in neoplastic and primary cells. An improvement in transient plasmid transfection is also seen, with as much as 85% of cells transiently expressing b-galactosidase (20-50 fold improvement). Stable transfection is only improved for linearized or nicked plasmids. Cells have improved gene transfer for at least 96 hours after irradiation. Both fractionated and continuous low dose rate irradiation are effective at improving stable gene transfer in mammalian cells, thus making relatively high radiation dose delivery clinically feasible. Inter-plasmid recombination is radiation dose dependent in nuclear extract assays, and the type of overhang (3', 5' or blunt end) significantly affects recombination efficiency and the type of product. The most common end-joining activity involves filling-in of the overhang followed by blunt end ligation. Adenovirus is a linear, double stranded DNA virus. We demonstrate that adenoviral infection efficiency is increased by irradiation. The duration of transgene expression is lengthened because the virus integrates with high efficiency (∼10

  6. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    International Nuclear Information System (INIS)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid; Gjerloev, Simon; Birk, Jesper; Roepke, Carsten; Norrild, Bodil

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induction of cell death. We have used the osteosarcoma cell line U2OS cells provided with E7 and the cdk2 inhibitor p21 (cip1/waf1) under inducible control, as a model system for the analysis of E7-mediated apoptosis. Our data shows that simultaneous expression of E7 and p21 proteins induces cell death, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge, this is the first time a role for cathepsin B is reported in HPV-induced apoptotic signalling

  7. Co-factor activated recombinant adenovirus proteinases

    Science.gov (United States)

    Anderson, Carl W.; Mangel, Walter F.

    1996-08-06

    This application describes methods and expression constructs for producing activatable recombinant adenovirus proteinases. Purified activatable recombinant adenovirus proteinases and methods of purification are described. Activated adenovirus proteinases and methods for obtaining activated adenovirus proteinases are further included. Isolated peptide cofactors of adenovirus proteinase activity, methods of purifying and identifying said peptide cofactors are also described. Antibodies immunoreactive with adenovirus proteinases, immunospecific antibodies, and methods for preparing them are also described. Other related methods and materials are also described.

  8. Simultaneous human papilloma virus type 16 E7 and cdk inhibitor p21 expression induces apoptosis and cathepsin B activation

    DEFF Research Database (Denmark)

    Kaznelson, Dorte Wissing; Bruun, Silas; Monrad, Astrid

    2004-01-01

    Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation and induc......Human papillomavirus type 16 (HPV-16) is the major risk factor for development of cervical cancer. The major oncoprotein E7 enhances cell growth control. However, E7 has in some reports been shown to induce apoptosis suggesting that there is a delicate balance between cell proliferation......, possibly because of conflicting growth control. Interestingly, E7/p21-induced cell death is associated with the activation of a newly identified mediator of apoptosis, namely cathepsin B. Activation of the cellular caspases is undetectable in cells undergoing E7/p21-induced apoptosis. To our knowledge...

  9. Aspartic cathepsin D degrades the cytosolic cysteine cathepsin inhibitor stefin B in the cells.

    Science.gov (United States)

    Železnik, Tajana Zajc; Kadin, Andrey; Turk, Vito; Dolenc, Iztok

    2015-09-18

    Stefin B is the major general cytosolic protein inhibitor of cysteine cathepsins. Its main function is to protect the organism against the activity of endogenous potentially hazardous proteases accidentally released from lysosomes. In this study, we investigated the possible effect of endosomal/lysosomal aspartic cathepsins D and E on stefin B after membrane permeabilization. Loss of membrane integrity of lysosomes and endosomes was induced by a lysosomotropic agent L-Leucyl-L-leucine methyl ester (Leu-Leu-OMe). The rat thyroid cell line FRTL-5 was selected as a model cell line owing to its high levels of proteases, including cathepsin D and E. Permeabilization of acid vesicles from FRTL-5 cells induced degradation of stefin B. The process was inhibited by pepstatin A, a potent inhibitor of aspartic proteases. However, degradation of stefin B was prevented by siRNA-mediated silencing of cathepsin D expression. In contrast, cathepsin E silencing had no effect on stefin B degradation. These results showed that cathepsin D and not cathepsin E degrades stefin B. It can be concluded that the presence of cathepsin D in the cytosol affects the inhibitory potency of stefin B, thus preventing the regulation of cysteine cathepsin activities in various biological processes. Copyright © 2015 Elsevier Inc. All rights reserved.

  10. E2-mediated cathepsin D (CTSD) activation involves looping of distal enhancer elements.

    Science.gov (United States)

    Bretschneider, Nancy; Kangaspeska, Sara; Seifert, Martin; Reid, George; Gannon, Frank; Denger, Stefanie

    2008-08-01

    Estrogen receptor alpha (ERalpha) is a ligand dependent transcription factor that regulates the expression of target genes through interacting with cis-acting estrogen response elements (EREs). However, only a minority of ERalpha binding sites are located within the proximal promoter regions of responsive genes. Here we report the characterization of an ERE located 9kbp upstream of the TSS of the cathepsin D gene (CTSD) that up-regulates CTSD expression upon estrogen stimulation in MCF-7 cells. Using ChIP, we show recruitment of ERalpha and phosphorylated PolII at the CTSD distal enhancer region. Moreover, we determine the kinetics of transient CpG methylation on the promoter region of CTSD and for the first time, at a distal enhancer element. We show that ERalpha is crucial for long-distance regulation of CTSD expression involving a looping mechanism.

  11. Molecular cloning and anti-invasive activity of cathepsin L propeptide-like protein from Calotropis procera R. Br. against cancer cells.

    Science.gov (United States)

    Kwon, Chang Woo; Yang, Hee; Yeo, SuBin; Park, Kyung-Min; Jeong, Ae Jin; Lee, Ki Won; Ye, Sang-Kyu; Chang, Pahn-Shick

    2018-12-01

    Cathepsin L of cancer cells has been shown to play an important role in degradation of extracellular matrix for metastasis. In order to reduce cell invasion, cathepsin L propeptide-like proteins which are classified as the I29 family in the MEROPS peptidase database were characterized from Calotropis procera R. Br., rich in cysteine protease. Of 19 candidates, the cloned and expressed recombinant SnuCalCp03-propeptide (rSnuCalCp03-propeptide) showed a low nanomolar K i value of 2.3 ± 0.2 nM against cathepsin L. A significant inhibition of tumor cell invasion was observed with H1975, HT29, MDA-BM-231, PANC1, and PC3 with a 76, 67, 67, 63, and 79% reduction, respectively, in invasion observed in the presence of 400 nM of the rSnuCalCp03-propeptide. In addition, thermal and pH study showed rSnuCalCp03-propeptide consisting of secondary structures was stable at a broad range of temperatures (30-70 °C) and pH (2-10, except for 5 which is close to the isoelectric point of 5.2).

  12. Activation of cathepsin L contributes to the irreversible depolarization induced by oxygen and glucose deprivation in rat hippocampal CA1 neurons.

    Science.gov (United States)

    Kikuta, Shogo; Murai, Yoshinaka; Tanaka, Eiichiro

    2017-01-01

    Oxygen and glucose deprivation (OGD) elicits a rapid and irreversible depolarization with a latency of ∼5min in intracellular recordings of hippocampal CA1 neurons in rat slice preparations. In the present study, we examined the role of cathepsin L in the OGD-induced depolarization. OGD-induced depolarizations were irreversible as no recovery of membrane potential was observed. The membrane potential reached 0mV when oxygen and glucose were reintroduced immediately after the onset of the OGD-induced rapid depolarization. The OGD-induced depolarizations became reversible when the slice preparations were pre-incubated with cathepsin L inhibitors (types I and IV at 0.3-2nM and 0.3-30nM, respectively). Moreover, pre-incubation with these cathepsin inhibitors prevented the morphological changes, including swelling of the cell soma and fragmentation of dendrites, observed in control neurons after OGD. These findings suggest that the activation of cathepsin L contributes to the irreversible depolarization produced by OGD. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    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

  14. Effect of mefenamic acid on the immunity and hemostatic system of cancer patients and on the activity of cathepsin D-like protease in colonic cancer tissue

    International Nuclear Information System (INIS)

    Klyachkin, B.M.; Basargin, S.T.; Timofeev, I.V.; Khaliulin, Yu.G.; Dorofeev, S.A.; Alekseenko, L.D.; Gumenyuk, M.L.

    1992-01-01

    The study of the effect of sodium mefenaminate on radiation resistance of mice yielded positive results. Clinical investigations showed mefenamic acid to decrease the activity of cathepsin D-like protease in colonic cancer tissue. The acid field to affect the proteolytic activity of the normal mucosa. It revealed an immunomodulating activity and influenced the hemostatic system which usually manifested itself in amelioration of hypercoagulation

  15. Collagenolytic activities of the major secreted cathepsin L peptidases involved in the virulence of the helminth pathogen, Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Mark W Robinson

    Full Text Available BACKGROUND: The temporal expression and secretion of distinct members of a family of virulence-associated cathepsin L cysteine peptidases (FhCL correlates with the entry and migration of the helminth pathogen Fasciola hepatica in the host. Thus, infective larvae traversing the gut wall secrete cathepsin L3 (FhCL3, liver migrating juvenile parasites secrete both FhCL1 and FhCL2 while the mature bile duct parasites, which are obligate blood feeders, secrete predominantly FhCL1 but also FhCL2. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that FhCL1, FhCL2 and FhCL3 exhibit differences in their kinetic parameters towards a range of peptide substrates. Uniquely, FhCL2 and FhCL3 readily cleave substrates with Pro in the P2 position and peptide substrates mimicking the repeating Gly-Pro-Xaa motifs that occur within the primary sequence of collagen. FhCL1, FhCL2 and FhCL3 hydrolysed native type I and II collagen at neutral pH but while FhCL1 cleaved only non-collagenous (NC, non-Gly-X-Y domains FhCL2 and FhCL3 exhibited collagenase activity by cleaving at multiple sites within the α1 and α2 triple helix regions (Col domains. Molecular simulations created for FhCL1, FhCL2 and FhCL3 complexed to various seven-residue peptides supports the idea that Trp67 and Tyr67 in the S2 subsite of the active sites of FhCL3 and FhCL2, respectively, are critical to conferring the unique collagenase-like activity to these enzymes by accommodating either Gly or Pro residues at P2 in the substrate. The data also suggests that FhCL3 accommodates hydroxyproline (Hyp-Gly at P3-P2 better than FhCL2 explaining the observed greater ability of FhCL3 to digest type I and II collagens compared to FhCL2 and why these enzymes cleave at different positions within the Col domains. CONCLUSIONS/SIGNIFICANCE: These studies further our understanding of how this helminth parasite regulates peptidase expression to ensure infection, migration and establishment in host tissues.

  16. Activated cathepsin L is associated with the switch from autophagy to apoptotic death of SH-SY5Y cells exposed to 6-hydroxydopamine

    Energy Technology Data Exchange (ETDEWEB)

    Li, Lingyun, E-mail: lingyunlee@126.com [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Gao, Luyan [Experimental Center, The Second Affiliated Hospital of Soochow University, Suzhou 215004 (China); Song, Yunzhen; Qin, Zheng-Hong [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China); Liang, Zhongqin, E-mail: liangzhongqin@suda.edu.cn [Department of Pharmacology, College of Pharmaceutical Sciences, Soochow University, Suzhou 215123 (China)

    2016-02-12

    Autophagy and apoptosis are common responses to pathological damage in the process of Parkinson's disease (PD), and lysosome dysfunction may contribute to the etiology of PD's neurodegenerative process. In this study, we demonstrated that the neurotoxin 6-hydroxydopamine (6-OHDA) increased autophagy in SH-SY5Y cells, as determined by detection of the lysosome marker lysosomal-associated membrane protein1, the autophagy protein light chain 3 (LC3)-II and the autophagy substrate P62 protein. Meanwhile, autophagy repression with 3-methyladenine accelerated the activation of caspase-3 and PARP and aggravated the cell apoptotic death induced by 6-OHDA. Furthermore, we found that 6-OHDA treatment resulted in a transient increase in the intracellular and nuclear expression of cathepsin L (CTSL). The CTSL inhibitor, Z-FY-CHO, could promote autophagy, decrease accumulation of P62, and block activation of caspase-3 and PARP. Taken together, these results suggest that activation of autophagy may primarily be a protective process in SH-SY5Y cell death induced by 6-OHDA, and the nuclear translocation of CTSL could enhance the cell apoptotic cascade via disturbing autophagy-apoptotic systems in SH-SY5Y cells. Our findings highlight the potential role of CTSL in the cross talk between autophagy and apoptosis, which might be considered a therapeutic strategy for treatment of pathologic conditions associated with neurodegeneration. - Highlights: • Inhibition of autophagy aggravated the cell apoptotic death in SH-SY5Y cells. • Activation of cathepsin L impaired the autophagy pathway. • Activation of cathepsin L enhanced the cell apoptotic cascade. • Cathepsin L involves in the cross talk between autophagy and apoptosis.

  17. Chemical constituents of the stem bark of Vochysia thyrsoidea Pohl. (Vochysiaceae) and evaluation of their cytotoxicity and inhibitory activity against cathepsins B and K

    International Nuclear Information System (INIS)

    Sousa, Lorena Ramos Freitas de; Silva, Jame's A. da; Vieira, Paulo Cezar; Costa, Maisa Borges; Santos, Mirley Luciene dos; Menezes, Antonio Carlos Severo; Sbardelotto, Aline Borba; Pessoa, Claudia do O; Moraes, Manoel Odorico de

    2014-01-01

    A new flavonoid, catechin-3-O-(3 - O-trans-cinnamoyl)-α-rhamnopyranoside, along with known compounds, catechin-3-O-α-rhamnopyranoside, 3-oxo-urs-12-en-28-oic acid, 2,4,6-trimethoxybenzoic acid, 2-butyl-D-fructofuranoside and 1-butyl-D-fructofuranoside, has been isolated from the stem bark of V. thyrsoidea. These compounds were assayed for inhibition of protease activity (cathepsins B and K) and against cancer cell lines. Catechin-3-O-(3 - O-trans-cinnamoyl)-α-rhamnopyranoside showed moderate inhibitory activity (IC 50 = 62.02 µM) against cathepsin B while 2-butyl-D-fructofuranoside was the most potent against a strain of CNS (SF-295) and human leukemia (HL-60) with IC 50 = 36.80 μM and IC 50 = 25.37 μM, respectively (author)

  18. Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise among limits of detection, reduced time, and resources.

    Science.gov (United States)

    Dumas, Jerald E; Platt, Manu O

    2013-07-01

    Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis, and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as clear bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 h to 10 min, incubation assay time from overnight to 4 h, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by Smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings.

  19. Systematic optimization of multiplex zymography protocol to detect active cathepsins K, L, S, and V in healthy and diseased tissue: compromise between limits of detection, reduced time, and resources

    Science.gov (United States)

    Dumas, Jerald E.; Platt, Manu O.

    2013-01-01

    Cysteine cathepsins are a family of proteases identified in cancer, atherosclerosis, osteoporosis, arthritis and a number of other diseases. As this number continues to rise, so does the need for low cost, broad use quantitative assays to detect their activity and can be translated to the clinic in the hospital or in low resource settings. Multiplex cathepsin zymography is one such assay that detects subnanomolar levels of active cathepsins K, L, S, and V in cell or tissue preparations observed as cleared bands of proteolytic activity after gelatin substrate SDS-PAGE with conditions optimal for cathepsin renaturing and activity. Densitometric analysis of the zymogram provides quantitative information from this low cost assay. After systematic modifications to optimize cathepsin zymography, we describe reduced electrophoresis time from 2 hours to 10 minutes, incubation assay time from overnight to 4 hours, and reduced minimal tissue protein necessary while maintaining sensitive detection limits; an evaluation of the pros and cons of each modification is also included. We further describe image acquisition by smartphone camera, export to Matlab, and densitometric analysis code to quantify and report cathepsin activity, adding portability and replacing large scale, darkbox imaging equipment that could be cost prohibitive in limited resource settings. PMID:23532386

  20. Elastin Degradation by Cathepsin V Requires Two Exosites*

    Science.gov (United States)

    Du, Xin; Chen, Nelson L. H.; Wong, Andre; Craik, Charles S.; Brömme, Dieter

    2013-01-01

    Cathepsin V is a highly effective elastase and has been implicated in physiological and pathological extracellular matrix degradation. However, its mechanism of action remains elusive. Whereas human cathepsin V exhibits a potent elastolytic activity, the structurally homologous cathepsin L, which shares a 78% amino acid sequence, has only a minimal proteolytic activity toward insoluble elastin. This suggests that there are distinct structural domains that play an important role in elastinolysis. In this study, a total of 11 chimeras of cathepsins V and L were generated to identify elastin-binding domains in cathepsin V. Evaluation of these chimeras revealed two exosites contributing to the elastolytic activity of cathepsin V that are distant from the active cleft of the protease and are located in surface loop regions. Replacement of exosite 1 or 2 with analogous residues from cathepsin L led to a 75 and 43% loss in the elastolytic activity, respectively. Replacement of both exosites yielded a non-elastase variant similar to that of cathepsin L. Identification of these exosites may contribute to the design of inhibitors that will only affect the elastolytic activity of cysteine cathepsins without interfering with other physiological protease functions. PMID:24121514

  1. Tumor marker utility and prognostic relevance of cathepsin B, cathepsin L, urokinase-type plasminogen activator, plasminogen activator inhibitor type-1, CEA and CA 19-9 in colorectal cancer

    International Nuclear Information System (INIS)

    Herszényi, László; Farinati, Fabio; Cardin, Romilda; István, Gábor; Molnár, László D; Hritz, István; De Paoli, Massimo; Plebani, Mario; Tulassay, Zsolt

    2008-01-01

    Cathepsin B and L (CATB, CATL), urokinase-type plasminogen activator (uPA) and its inhibitor PAI-1 play an important role in colorectal cancer invasion. The tumor marker utility and prognostic relevance of these proteases have not been evaluated in the same experimental setting and compared with that of CEA and CA-19-9. Protease, CEA and CA 19-9 serum or plasma levels were determined in 56 patients with colorectal cancer, 25 patients with ulcerative colitis, 26 patients with colorectal adenomas and 35 tumor-free control patients. Protease, CEA, CA 19-9 levels have been determined by ELISA and electrochemiluminescence immunoassay, respectively; their sensitivity, specificity, diagnostic accuracy have been calculated and correlated with clinicopathological staging. The protease antigen levels were significantly higher in colorectal cancer compared with other groups. Sensitivity of PAI-1 (94%), CATB (82%), uPA (69%), CATL (41%) were higher than those of CEA or CA 19-9 (30% and 18%, respectively). PAI-1, CATB and uPA demonstrated a better accuracy than CEA or CA 19-9. A combination of PAI-1 with CATB or uPA exhibited the highest sensitivity value (98%). High CATB, PAI-1, CEA and CA 19-9 levels correlated with advanced Dukes stages. CATB (P = 0.0004), CATL (P = 0.02), PAI-1 (P = 0.01) and CA 19-9 (P = 0.004) had a significant prognostic impact. PAI-1 (P = 0.001), CATB (P = 0.04) and CA 19-9 (P = 0.02) proved as independent prognostic variables. At the time of clinical detection proteases are more sensitive indicators for colorectal cancer than the commonly used tumor markers. Determinations of CATB, CATL and PAI-1 have a major prognostic impact in patients with colorectal cancer

  2. Anti-proliferative activity of recombinant melittin expressed in ...

    African Journals Online (AJOL)

    Recombinant melittin was then successfully expressed in Escherichia coli. The activity of affinity-purified recombinant melittin was determined in human leukemic U937 cells. Results show that the recombinant melittin had the same anti-proliferative activity in human leukemic U937 cells in vitro as natural one. This shows the ...

  3. Cathepsin D, a Marker for the Metastatic Potential of Breast Cancer, May Regulate the Mitogenic Activity of Fibroblast Growth Factor 1

    National Research Council Canada - National Science Library

    Grieb, Teri

    1998-01-01

    .... Over the years, the data substantiating such a role for cathepsin D has been quite conflicting However, there is strong evidence that cathepsin D plays a role in the degradation of the extracellular matrix (ECM...

  4. Cathepsin E promotes pulmonary emphysema via mitochondrial fission.

    Science.gov (United States)

    Zhang, Xuchen; Shan, Peiying; Homer, Robert; Zhang, Yi; Petrache, Irina; Mannam, Praveen; Lee, Patty J

    2014-10-01

    Emphysema is characterized by loss of lung elasticity and irreversible air space enlargement, usually in the later decades of life. The molecular mechanisms of emphysema remain poorly defined. We identified a role for a novel cathepsin, cathepsin E, in promoting emphysema by inducing mitochondrial fission. Unlike previously reported cysteine cathepsins, which have been implicated in cigarette smoke-induced lung disease, cathepsin E is a nonlysosomal intracellular aspartic protease whose function has been described only in antigen processing. We examined lung tissue sections of persons with chronic obstructive pulmonary disease, a clinical entity that includes emphysematous change. Human chronic obstructive pulmonary disease lungs had markedly increased cathepsin E protein in the lung epithelium. We generated lung epithelial-targeted transgenic cathepsin E mice and found that they develop emphysema. Overexpression of cathepsin E resulted in increased E3 ubiquitin ligase parkin, mitochondrial fission protein dynamin-related protein 1, caspase activation/apoptosis, and ultimately loss of lung parenchyma resembling emphysema. Inhibiting dynamin-related protein 1, using a small molecule inhibitor in vitro or in vivo, inhibited cathepsin E-induced apoptosis and emphysema. To the best of our knowledge, our study is the first to identify links between cathepsin E, mitochondrial fission, and caspase activation/apoptosis in the pathogenesis of pulmonary emphysema. Our data expand the current understanding of molecular mechanisms of emphysema development and may provide new therapeutic targets. Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

  5. Aspartic cathepsin D endopeptidase contributes to extracellular digestion in clawed lobsters Homarus americanus and Homarus gammarus.

    Science.gov (United States)

    Rojo, Liliana; Muhlia-Almazan, Adriana; Saborowski, Reinhard; García-Carreño, Fernando

    2010-11-01

    Acid digestive proteinases were studied in the gastric fluids of two species of clawed lobster (Homarus americanus and Homarus gammarus). An active protein was identified in both species as aspartic proteinase by specific inhibition with pepstatin A. It was confirmed as cathepsin D by mass mapping, N-terminal, and full-length cDNA sequencing. Both lobster species transcribed two cathepsin D mRNAs: cathepsin D1 and cathepsin D2. Cathepsin D1 mRNA was detected only in the midgut gland, suggesting its function as a digestive enzyme. Cathepsin D2 mRNA was found in the midgut gland, gonads, and muscle. The deduced amino acid sequence of cathepsin D1 and cathepsin D2 possesses two catalytic DTG active-site motifs, the hallmark of aspartic proteinases. The putatively active cathepsin D1 has a molecular mass of 36.4 kDa and a calculated pI of 4.14 and possesses three potential glycosylation sites. The sequences showed highest similarities with cathepsin D from insects but also with another crustacean cathepsin D. Cathepsin D1 transcripts were quantified during a starvation period using real-time qPCR. In H. americanus, 15 days of starvation did not cause significant changes, but subsequent feeding caused a 2.5-fold increase. In H. gammarus, starvation caused a 40% reduction in cathepsin D1 mRNA, and no effect was observed with subsequent feeding.

  6. A bioavailable cathepsin S nitrile inhibitor abrogates tumor development.

    Science.gov (United States)

    Wilkinson, Richard D A; Young, Andrew; Burden, Roberta E; Williams, Rich; Scott, Christopher J

    2016-04-21

    Cathepsin S has been implicated in a variety of malignancies with genetic ablation studies demonstrating a key role in tumor invasion and neo-angiogenesis. Thus, the application of cathepsin S inhibitors may have clinical utility in the treatment of cancer. In this investigation, we applied a cell-permeable dipeptidyl nitrile inhibitor of cathepsin S, originally developed to target cathepsin S in inflammatory diseases, in both in vitro and in vivo tumor models. Validation of cathepsin S selectivity was carried out by assaying fluorogenic substrate turnover using recombinant cathepsin protease. Complete kinetic analysis was carried out and true K i values calculated. Abrogation of tumour invasion using murine MC38 and human MCF7 cell lines were carried out in vitro using a transwell migration assay. Effect on endothelial tube formation was evaluated using primary HUVEC cells. The effect of inhibitor in vivo on MC38 and MCF7 tumor progression was evaluated using cells propagated in C57BL/6 and BALB/c mice respectively. Subsequent immunohistochemical staining of proliferation (Ki67) and apoptosis (TUNEL) was carried out on MCF7 tumors. We confirmed that this inhibitor was able to selectively target cathepsin S over family members K, V, L and B. The inhibitor also significantly reduced MC38 and MCF7 cell invasion and furthermore, significantly reduced HUVEC endothelial tubule formation in vitro. In vivo analysis revealed that the compound could significantly reduce tumor volume in murine MC38 syngeneic and MCF7 xenograft models. Immunohistochemical analysis of MCF7 tumors revealed cathepsin S inhibitor treatment significantly reduced proliferation and increased apoptosis. In summary, these results highlight the characterisation of this nitrile cathepsin S inhibitor using in vitro and in vivo tumor models, presenting a compound which may be used to further dissect the role of cathepsin S in cancer progression and may hold therapeutic potential.

  7. Dissecting the active site of the collagenolytic cathepsin L3 protease of the invasive stage of Fasciola hepatica.

    Directory of Open Access Journals (Sweden)

    Ileana Corvo

    Full Text Available A family of secreted cathepsin L proteases with differential activities is essential for host colonization and survival in the parasitic flatworm Fasciola hepatica. While the blood feeding adult secretes predominantly FheCL1, an enzyme with a strong preference for Leu at the S2 pocket of the active site, the infective stage produces FheCL3, a unique enzyme with collagenolytic activity that favours Pro at P2.Using a novel unbiased multiplex substrate profiling and mass spectrometry methodology (MSP-MS, we compared the preferences of FheCL1 and FheCL3 along the complete active site cleft and confirm that while the S2 imposes the greatest influence on substrate selectivity, preferences can be indicated on other active site subsites. Notably, we discovered that the activity of FheCL1 and FheCL3 enzymes is very different, sharing only 50% of the cleavage sites, supporting the idea of functional specialization. We generated variants of FheCL1 and FheCL3 with S2 and S3 residues by mutagenesis and evaluated their substrate specificity using positional scanning synthetic combinatorial libraries (PS-SCL. Besides the rare P2 Pro preference, FheCL3 showed a distinctive specificity at the S3 pocket, accommodating preferentially the small Gly residue. Both P2 Pro and P3 Gly preferences were strongly reduced when Trp67 of FheCL3 was replaced by Leu, rendering the enzyme incapable of digesting collagen. In contrast, the inverse Leu67Trp substitution in FheCL1 only slightly reduced its Leu preference and improved Pro acceptance in P2, but greatly increased accommodation of Gly at S3.These data reveal the significance of S2 and S3 interactions in substrate binding emphasizing the role for residue 67 in modulating both sites, providing a plausible explanation for the FheCL3 collagenolytic activity essential to host invasion. The unique specificity of FheCL3 could be exploited in the design of specific inhibitors selectively directed to specific infective stage

  8. Highly Conserved Arg Residue of ERFNIN Motif of Pro-Domain is Important for pH-Induced Zymogen Activation Process in Cysteine Cathepsins K and L.

    Science.gov (United States)

    Aich, Pulakesh; Biswas, Sampa

    2018-06-01

    Pro-domain of a cysteine cathepsin contains a highly conserved Ex 2 Rx 2 Fx 2 Nx 3 Ix 3 N (ERFNIN) motif. The zymogen structure of cathepsins revealed that the Arg(R) residue of the motif is a central residue of a salt-bridge/H-bond network, stabilizing the scaffold of the pro-domain. Importance of the arginine is also demonstrated in studies where a single mutation (Arg → Trp) in human lysosomal cathepsin K (hCTSK) is linked to a bone-related genetic disorder "Pycnodysostosis". In the present study, we have characterized in vitro Arg → Trp mutant of hCTSK and the same mutant of hCTSL. The R → W mutant of hCTSK revealed that this mutation leads to an unstable zymogen that is spontaneously activated and auto-proteolytically degraded rapidly. In contrast, the same mutant of hCTSL is sufficiently stable and has proteolytic activity almost like its wild-type counterpart; however it shows an altered zymogen activation condition in terms of pH, temperature and time. Far and near UV circular dichroism and intrinsic tryptophan fluorescence experiments have revealed that the mutation has minimal effect on structure of the protease hCTSL. Molecular modeling studies shows that the mutated Trp31 in hCTSL forms an aromatic cluster with Tyr23 and Trp30 leading to a local stabilization of pro-domain and supplements the loss of salt-bridge interaction mediated by Arg31 in wild-type. In hCTSK-R31W mutant, due to presence of a non-aromatic Ser30 residue such interaction is not possible and may be responsible for local instability. These differences may cause detrimental effects of R31W mutation on the regulation of hCTSK auto-activation process compared to altered activation process in hCTSL.

  9. Histopathological analysis of cellular localization of cathepsins in abdominal aortic aneurysm wall.

    Science.gov (United States)

    Lohoefer, Fabian; Reeps, Christian; Lipp, Christina; Rudelius, Martina; Zimmermann, Alexander; Ockert, Stefan; Eckstein, Hans-Henning; Pelisek, Jaroslav

    2012-08-01

    An important feature of abdominal aortic aneurysm (AAA) is the destruction of vessel wall, especially elastin and collagen. Besides matrix metalloproteinases, cathepsins are the most potent elastolytic enzymes. The expression of cathepsins with known elastolytic and collagenolytic activities in the individual cells within AAA has not yet been determined. The vessel wall of 32 AAA patients and 10 organ donors was analysed by immunohistochemistry for expression of cathepsins B, D, K, L and S, and cystatin C in all cells localized within AAA. Luminal endothelial cells (ECs) of AAA were positive for cathepsin D and partially for cathepsins B, K and S. Endothelial cells of the neovessels and smooth muscle cells in the media were positive for all cathepsins tested, especially for cathepsin B. In the inflammatory infiltrate all cathepsins were expressed in the following pattern: B > D = S > K = L. Macrophages showed the highest staining intensity for all cathepsins. Furthermore, weak overall expression of cystatin C was observed in all the cells localized in the AAA with the exception of the ECs. There is markedly increased expression of the various cathepsins within the AAA wall compared to healthy aorta. Our data are broadly consistent with a role for cathepsins in AAA; and demonstrate expression of cathepsins D, B and S in phagocytic cells in the inflammatory infiltrate; and also may reveal a role for cathepsin B in lymphocytes. © 2012 The Authors. International Journal of Experimental Pathology © 2012 International Journal of Experimental Pathology.

  10. Expression of Cathepsins B, D, and G in Isocitrate Dehydrogenase-Wildtype Glioblastoma

    Directory of Open Access Journals (Sweden)

    Sabrina P. Koh

    2017-05-01

    Full Text Available AimTo investigate the expression of cathepsins B, D, and G, in relation to the cancer stem cell (CSC subpopulations, we have previously characterized within isocitrate dehydogenase (IDH-wildtype glioblastoma (IDHWGB.Methods3,3-Diaminobezidine (DAB immunohistochemical (IHC staining for cathepsins B, D, and G, was performed on 4μm-thick formalin-fixed paraffin-embedded IDHWGB samples obtained from six patients. Two representative DHWGB samples from the original cohort of patients were selected for immunofluorescent (IF IHC staining, to identify the localization of the cathepsins in relation to the CSC subpopulations. NanoString gene expression analysis and colorimetric in situ hybridization (CISH were conducted to investigate the transcriptional activation of genes encoding for cathepsins B, D, and G. Data obtained from cell counting of DAB IHC-stained slides and from NanoString analysis were subjected to statistical analyses to determine significance.ResultsCathepsin B and cathepsin D were detected in IDHWGB by DAB IHC staining. IF IHC staining demonstrated the expression of both cathepsin B and cathepsin D by the OCT4+ and SALL4+ CSC subpopulations. NanoString gene analysis and CISH confirmed the abundant transcript expression of these cathepsins. The transcriptional and translational expressions of cathepsin G were minimal and were confined to cells within the microvasculature.ConclusionThis study demonstrated the expression of cathepsin B and cathepsin D but not cathepsin G within the CSC subpopulations of IDHWGB at both the transcriptional and translational level. Cathepsin G was expressed at low levels and was not localized to the CSC population of IDHWGB. The novel finding of cathepsin B and cathepsin D in IDHWGB suggests the presence of bypass loops for the renin-angiotensin system, which may facilitate the production of angiotensin peptides. Elucidating the precise role of these cathepsins may lead to better understanding and more

  11. The Feasibility of Enzyme Targeted Activation for Amino Acid/Dipeptide Monoester Prodrugs of Floxuridine; Cathepsin D as a Potential Targeted Enzyme

    Directory of Open Access Journals (Sweden)

    Gordon L. Amidon

    2012-03-01

    Full Text Available The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5¢-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0–105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5¢-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine and 5¢-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enyzmes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5¢-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

  12. The feasibility of enzyme targeted activation for amino acid/dipeptide monoester prodrugs of floxuridine; cathepsin D as a potential targeted enzyme.

    Science.gov (United States)

    Tsume, Yasuhiro; Amidon, Gordon L

    2012-03-26

    The improvement of therapeutic efficacy for cancer agents has been a big challenge which includes the increase of tumor selectivity and the reduction of adverse effects at non-tumor sites. In order to achieve those goals, prodrug approaches have been extensively investigated. In this report, the potential activation enzymes for 5'-amino acid/dipeptide monoester floxuridine prodrugs in pancreatic cancer cells were selected and the feasibility of enzyme specific activation of prodrugs was evaluated. All prodrugs exhibited the range of 3.0-105.7 min of half life in Capan-2 cell homogenate with the presence and the absence of selective enzyme inhibitors. 5'-O-L-Phenylalanyl-L-tyrosyl-floxuridine exhibited longer half life only with the presence of pepstatin A. Human cathepsin B and D selectively hydrolized 5'-O-L-phenylalanyl-L-tyrosylfloxuridine and 5'-O-L-phenylalanyl-L-glycylfloxuridine compared to the other tested prodrugs. The wide range of growth inhibitory effect by floxuridine prodrugs in Capan-2 cells was observed due to the different affinities of prodrug promoieties to enzymes. In conclusion, it is feasible to design prodrugs which are activated by specific enzymes. Cathepsin D might be a good candidate as a target enzyme for prodrug activation and 5'-O-L-phenylalanyl-L-tyrosylfloxuridine may be the best candidate among the tested floxuridine prodrugs.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

  14. Recombinant ArtinM activates mast cells.

    Science.gov (United States)

    Barbosa-Lorenzi, Valéria Cintra; Cecilio, Nerry Tatiana; de Almeida Buranello, Patricia Andressa; Pranchevicius, Maria Cristina; Goldman, Maria Helena S; Pereira-da-Silva, Gabriela; Roque-Barreira, Maria Cristina; Jamur, Maria Célia; Oliver, Constance

    2016-07-04

    Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing β-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.

  15. Detection of femtomole quantities of mature cathepsin K with zymography.

    Science.gov (United States)

    Li, Weiwei A; Barry, Zachary T; Cohen, Joshua D; Wilder, Catera L; Deeds, Rebecca J; Keegan, Philip M; Platt, Manu O

    2010-06-01

    Cathepsin K, the most potent mammalian collagenase, has been implicated in osteoporosis, cancer metastasis, atherosclerosis, and arthritis. Although procathepsin K is stable and readily detected, the active mature cathepsin K eludes detection by in vitro methods due to its shorter half-life and inactivation at neutral pH. We describe, for the first time, reliable detection, visualization, and quantification of mature cathepsin K to femtomole resolution using gelatin zymography. The specificity of the method was validated with cathepsin K knockdown using small interfering RNA (siRNA) transfection of human monocyte-derived macrophages, and enzymatic activity confirmed with benzyloxycarbonyl-glycine-proline-arginine-7-amino-4-methylcoumarin (Z-GPR-AMC) substrate hydrolysis was fit to a computational model of enzyme kinetics. Furthermore, cathepsin K zymography was used to show that murine osteoclasts secrete more cathepsin K than is stored intracellularly, and this was opposite to the behavior of the macrophages from which they were differentiated. In summary, this inexpensive, species-independent, antibody-free protocol describes a sensitive method with broad potential to elucidate previously undetectable cathepsin K activity. Copyright 2010 Elsevier Inc. All rights reserved.

  16. Cathepsin G-dependent modulation of platelet thrombus formation in vivo by blood neutrophils.

    Directory of Open Access Journals (Sweden)

    Nauder Faraday

    Full Text Available Neutrophils are consistently associated with arterial thrombotic morbidity in human clinical studies but the causal basis for this association is unclear. We tested the hypothesis that neutrophils modulate platelet activation and thrombus formation in vivo in a cathepsin G-dependent manner. Neutrophils enhanced aggregation of human platelets in vitro in dose-dependent fashion and this effect was diminished by pharmacologic inhibition of cathepsin G activity and knockdown of cathepsin G expression. Tail bleeding time in the mouse was prolonged by a cathepsin G inhibitor and in cathepsin G knockout mice, and formation of neutrophil-platelet conjugates in blood that was shed from transected tails was reduced in the absence of cathepsin G. Bleeding time was highly correlated with blood neutrophil count in wildtype but not cathepsin G deficient mice. In the presence of elevated blood neutrophil counts, the anti-thrombotic effect of cathepsin G inhibition was greater than that of aspirin and additive to it when administered in combination. Both pharmacologic inhibition of cathepsin G and its congenital absence prolonged the time for platelet thrombus to form in ferric chloride-injured mouse mesenteric arterioles. In a vaso-occlusive model of ischemic stroke, inhibition of cathepsin G and its congenital absence improved cerebral blood flow, reduced histologic brain injury, and improved neurobehavioral outcome. These experiments demonstrate that neutrophil cathepsin G is a physiologic modulator of platelet thrombus formation in vivo and has potential as a target for novel anti-thrombotic therapies.

  17. Mycobacterium tuberculosis Modulates miR-106b-5p to Control Cathepsin S Expression Resulting in Higher Pathogen Survival and Poor T-Cell Activation

    Directory of Open Access Journals (Sweden)

    David Pires

    2017-12-01

    Full Text Available The success of tuberculosis (TB bacillus, Mycobacterium tuberculosis (Mtb, relies on the ability to survive in host cells and escape to immune surveillance and activation. We recently demonstrated that Mtb manipulation of host lysosomal cathepsins in macrophages leads to decreased enzymatic activity and pathogen survival. In addition, while searching for microRNAs (miRNAs involved in posttranscriptional gene regulation during mycobacteria infection of human macrophages, we found that selected miRNAs such as miR-106b-5p were specifically upregulated by pathogenic mycobacteria. Here, we show that miR-106b-5p is actively manipulated by Mtb to ensure its survival in macrophages. Using an in silico prediction approach, we identified miR-106b-5p with a potential binding to the 3′-untranslated region of cathepsin S (CtsS mRNA. We demonstrated by luminescence-based methods that miR-106b-5p indeed targets CTSS mRNA resulting in protein translation silencing. Moreover, miR-106b-5p gain-of-function experiments lead to a decreased CtsS expression favoring Mtb intracellular survival. By contrast, miR-106b-5p loss-of-function in infected cells was concomitant with increased CtsS expression, with significant intracellular killing of Mtb and T-cell activation. Modulation of miR-106b-5p did not impact necrosis, apoptosis or autophagy arguing that miR-106b-5p directly targeted CtsS expression as a way for Mtb to avoid exposure to degradative enzymes in the endocytic pathway. Altogether, our data suggest that manipulation of miR-106b-5p as a potential target for host-directed therapy for Mtb infection.

  18. Cathepsins are required for Toll-like receptor 9 responses

    International Nuclear Information System (INIS)

    Matsumoto, Fumi; Saitoh, Shin-ichiroh; Fukui, Ryutaroh; Kobayashi, Toshihiko; Tanimura, Natsuko; Konno, Kazunori; Kusumoto, Yutaka; Akashi-Takamura, Sachiko; Miyake, Kensuke

    2008-01-01

    Toll-like receptors (TLR) recognize a variety of microbial products and activate defense responses. Pathogen sensing by TLR2/4 requires accessory molecules, whereas little is known about a molecule required for DNA recognition by TLR9. After endocytosis of microbes, microbial DNA is exposed and recognized by TLR9 in lysosomes. We here show that cathepsins, lysosomal cysteine proteases, are required for TLR9 responses. A cell line Ba/F3 was found to be defective in TLR9 responses despite enforced TLR9 expression. Functional cloning with Ba/F3 identified cathepsin B/L as a molecule required for TLR9 responses. The protease activity was essential for the complementing effect. TLR9 responses were also conferred by cathepsin S or F, but not by cathepsin H. TLR9-dependent B cell proliferation and CD86 upregulation were apparently downregulated by cathepsin B/L inhibitors. Cathepsin B inhibitor downregulated interaction of CpG-B with TLR9 in 293T cells. These results suggest roles for cathepsins in DNA recognition by TLR9

  19. The highly antigenic 53/25 kDa Taenia solium protein fraction with cathepsin-L like activity is present in the oncosphere/cysticercus and induces non-protective IgG antibodies in pigs

    Science.gov (United States)

    Zimic, Mirko; Pajuelo, Mónica; Gilman, Robert H.; Gutiérrez, Andrés H.; Rueda, Luis D.; Flores, Myra; Chile, Nancy; Verástegui, Manuela; Gonzalez, Armando; García, Héctor H.; Sheen, Patricia

    2011-01-01

    Cathepsin L-like proteases are secreted by several parasites including Taenia solium. The mechanism used by T. solium oncospheres to degrade and penetrate the intestine and infect the host is incompletely understood. It is assumed that intestinal degradation is driven by the proteolytic activity of enzymes secreted by the oncosphere. Blocking the proteolytic activity by an antibody response would prevent the oncosphere penetration and further infection. Serine and cysteine proteases including chymotrypsin, trypsin, elastase, and cathepsin L, are secreted by T. solium and Taenia saginata oncospheres when cultured in vitro, being potential vaccine candidates. However, the purification of a sufficient quantity of proteases secreted by oncospheres to conduct a vaccine trial is costly and lengthy. A 53/25 kDa cathepsin L-like fraction partially purified from T. solium cyst fluid was described previously as an important antigen for immunodiagnostics. In this study we found that this antigen is present in the T. solium oncosphere and is also secreted by the cysticercus. This protein fraction was tested for its ability to protect pigs against an oral challenge with T. solium oncospheres in a vaccine trial. IgG antibodies against the 53/25 kDa cathepsin L-like protein fraction were elicited in the vaccinated animals but did not confer protection. PMID:22119017

  20. Activity of recombinant factor VIIa under different conditions in vitro

    DEFF Research Database (Denmark)

    Bladbjerg, Else-Marie; Jespersen, Jørgen

    2008-01-01

    Recombinant activated factor VII (NovoSeven; Novo Nordisk A/S, Måløv, Denmark) is an effective drug for treatment of bleeding in patients with haemophilia A or B and inhibitors. Little is known about physiological conditions influencing the efficacy of recombinant activated factor VII. We...... investigated the in-vitro effects of pH, temperature, and haemodilution on the activity of recombinant activated factor VII. Samples from eight healthy volunteers were spiked with recombinant activated factor VII (final concentration 1.7 microg/ml) and adjusted to pH 6.0, 6.5, 7.0, and 7.4 or analysed at 30......, 33, 37, and 40 degrees C, or diluted 0, 10, 20, 40, and 60% with dextran before analysis. Samples were analysed as rotational thromboelastometry in whole blood (clotting time, clot formation time, and maximum clot firmness) with and without Innovin (tissue factor), and as factor VII coagulant...

  1. Recombiner

    International Nuclear Information System (INIS)

    Kikuchi, Nobuo.

    1983-01-01

    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.)

  2. Recombiner

    International Nuclear Information System (INIS)

    Osumi, Morimichi.

    1979-01-01

    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.)

  3. Acid-Mediated Tumor Proteolysis: Contribution of Cysteine Cathepsins

    Directory of Open Access Journals (Sweden)

    Jennifer M Rothberg

    2013-10-01

    Full Text Available One of the noncellular microenvironmental factors that contribute to malignancy of solid tumors is acidic peritumoral pH. We have previously demonstrated that extracellular acidosis leads to localization of the cysteine pro-tease cathepsin B on the tumor cell membrane and its secretion. The objective of the present study was to determine if an acidic extracellular pH such as that observed in vivo (i.e., pHe 6.8 affects the activity of proteases, e.g., cathepsin B, that contribute to degradation of collagen IV by tumor cells when grown in biologically relevant three-dimensional (3D cultures. For these studies, we used 1 3D reconstituted basement membrane overlay cultures of human carcinomas, 2 live cell imaging assays to assess proteolysis, and 3 in vivo imaging of active tumor proteases. At pHe 6.8, there were increases in pericellular active cysteine cathepsins and in degradation of dye-quenched collagen IV, which was partially blocked by a cathepsin B inhibitor. Imaging probes for active cysteine cathepsins localized to tumors in vivo. The amount of bound probe decreased in tumors in bicarbonate-treated mice, a treatment previously shown to increase peritumoral pHe and reduce local invasion of the tumors. Our results are consistent with the acid-mediated invasion hypothesis and with a role for cathepsin B in promoting degradation of a basement membrane protein substrate, i.e., type IV collagen, in an acidic peritumoral environment.

  4. Prdm9 Controls Activation of Mammalian Recombination Hotspots

    OpenAIRE

    Parvanov, Emil D.; Petkov, Petko M.; Paigen, Kenneth

    2009-01-01

    Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, assures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse Chr 17, that controls activation of recombination at multiple distant hotspots, has been mapped within a 181 Kb interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is uniquel...

  5. The 3D structure and function of digestive cathepsin L-like proteinases of Tenebrio molitor larval midgut.

    Science.gov (United States)

    Beton, Daniela; Guzzo, Cristiane R; Ribeiro, Alberto F; Farah, Chuck S; Terra, Walter R

    2012-09-01

    Cathepsin L-like proteinases (CAL) are major digestive proteinases in the beetle Tenebrio molitor. Procathepsin Ls 2 (pCAL2) and 3 (pCAL3) were expressed as recombinant proteins in Escherichia coli, purified and activated under acidic conditions. Immunoblot analyses of different T. molitor larval tissues demonstrated that a polyclonal antibody to pCAL3 recognized pCAL3 and cathepsin L 3 (CAL3) only in the anterior two-thirds of midgut tissue and midgut luminal contents of T. molitor larvae. Furthermore, immunocytolocalization data indicated that pCAL3 occurs in secretory vesicles and microvilli in anterior midgut. Therefore CAL3, like cathepsin L 2 (CAL2), is a digestive enzyme secreted by T. molitor anterior midgut. CAL3 hydrolyses Z-FR-MCA and Z-RR-MCA (typical cathepsin substrates), whereas CAL2 hydrolyses only Z-FR-MCA. Active site mutants (pCAL2C25S and pCAL3C26S) were constructed by replacing the catalytic cysteine with serine to prevent autocatalytic processing. Recombinant pCAL2 and pCAL3 mutants (pCAL2C25S and pCAL3C26S) were prepared, crystallized and their 3D structures determined at 1.85 and 2.1 Å, respectively. While the overall structure of these enzymes is similar to other members of the papain superfamily, structural differences in the S2 subsite explain their substrate specificities. The data also supported models for CAL trafficking to lysosomes and to secretory vesicles to be discharged into midgut contents. Copyright © 2012 Elsevier Ltd. All rights reserved.

  6. Cathepsin L in human meningiomas

    International Nuclear Information System (INIS)

    Trinkaus, M.; Lah, T.T.; Vranic, A.; Dolenc, V.V.

    2003-01-01

    Background. Although meningiomas are considered as benign tumours, about 10% comprise a subgroup of a typical meningiomas, classified as WHO grade II, with greater likelihood of recurrences and/or aggressive behaviour, including the possibility of brain tissue invasion. The lysosomal cysteine endopeptidase cathepsin L plays a role in tumour cell invasion and malignant progression of cancer, and has been suggested as a prognostic marker for certain types of tumours. Results. In our study, we compared the expression of cathepsin L in 30 meningiomas with their clinical invasiveness. Cathepsin L was determined by immunohistochemical analysis, quantitative real-time RT-PCR and Northern blot. We showed that expression of cathepsin L protein was significantly higher (p=0.019) in 9 atypical than in 21 benign meningiomas. Within the group of benign meningiomas, expression of cathepsin L was significantly lower in the transitional histological subtype. We measured the levels of cathepsin LA type of RNA splicing variants: LA, LAI and LAII, but not LAIII and not the LB variant, the latter being several times lower than the LA type. In contrast to protein levels, the levels of cathepsin LA, AI, AII RNA variants did not differ between histological subtypes or between benign and a typical meningiomas. The expression of total measured cathepsin LA, AI, AII RNA variants in the samples, taken from the centre and the periphery of the tumours, also showed no statistically significant differences. Conclusions. These results indicate that cathepsin L protein over-expression may contribute to the development of the aggressive and possibly invasive character of a typical meningiomas and that it may be up regulated at the translational level. (author)

  7. Cytosol cathepsin-D content and proliferative activity of human breast cancer. The Comitato Italiano per il Controllo di Qualita del Laboratorio in Oncologia.

    Science.gov (United States)

    Paradiso, A; Mangia, A; Correale, M; Abbate, I; Ferri, G; Piffanelli, A; Catozzi, L; Amadori, D; Riccobon, A; De Lena, M

    1992-01-01

    Mitogenic properties have been demonstrated in vitro for the lysosomal acidic protease cathepsin-D (cath-D). We investigated possible relationships between cath-D cytosol cell content and tumor proliferative activity in a series of 129 operable breast cancer patients. For total cytosol cath-D evaluation, a solid phase two-site immunoradiometric assay was utilized on tumor cell cytosol obtained for hormone receptor assay (DCC method). The percentage of S-phase cells was analyzed by 3H-thymidine autoradiographic assay. Median 3H-thymidine Labeling Index (3H-Tdr-LI) of the series was 2.7%; median cath-D content resulted 57 pmol/mg of protein cytosol and was significantly higher in node-positive with respect to the node-negative subgroup (p < 0.03). When classified in low, intermediate or high tumor cath-D content and slow or fast proliferative activity (cut-off: median values of the series), no significant agreement was found between the two variables. Statistical analysis, however, showed that a significant inverse correlation existed in node positive tumors between cath-D and 3H-Tdr-LI values which was even more evident in N-positive high estrogen receptor-positive (ER+) cases (coefficient of correlation = 0.6828; p = 0.0001). Cytosol cath-D content cannot be generally proposed as a direct marker of proliferative activity for operable breast cancer.

  8. Antiproliferative activity of recombinant human interferon-λ2 ...

    African Journals Online (AJOL)

    Antiproliferative activity of recombinant human interferon-λ2 expressed in stably ... The representing 26 kDa protein band of IFN-λ2 was detected by SDS-PAGE and ... The antiproliferative activity of hIFN-λ2 was determined by MTT assay.

  9. Tamm-Horsfall Glycoprotein Enhances PMN Phagocytosis by Binding to Cell Surface-Expressed Lactoferrin and Cathepsin G That Activates MAP Kinase Pathway

    Directory of Open Access Journals (Sweden)

    Chia-Li Yu

    2011-03-01

    Full Text Available The molecular basis of polymorphonuclear neutrophil (PMN phagocytosis-enhancing activity (PEA by human purified urinary Tamm-Horsfall glyco- protein (THP has not been elucidated. In this study, we found human THP bound to lactoferrin (LF and cathepsin G (CG expressed on the surface of PMN, identified by a proteomic study with MALDI-TOF- LC/LC/mass spectrometric analysis. Pre-incubation of 10% SDS-PAGE electrophoresed PMN lysates with monoclonal anti-LF or anti-CG antibody reduced the binding with THP. To elucidate the signaling pathway of THP on PMN activation, we found THP enhanced ERK1/2 phosphorylation, reduced p38 MAP kinase phosphorylation, but had no effect on DNA binding of the five NF-kB family members in PMN. To further clarify whether the carbohydrate-side chains or protein-core structure in THP molecule is responsible for THP-PEA, THP was cleaved by different degrading enzymes with carbohydrate specificity (neuraminidase and β-galactosidase, protein specificity (V8 protease and proteinase K or glycoconjugate specificity (carboxylpeptidase Y and O-sialoglycoprotein endopeptidase. We clearly demonstrated that the intact protein-core structure in THP molecule was more important for THP-PEA than carbohydrate-side chains. Putting these results together, we conclude that THP adheres to surface-expressed LF and CG on PMN and transduces signaling via the MAP kinase pathway to enhance PMN phagocytosis.

  10. Cysteine Cathepsins as Regulators of the Cytotoxicity of NK and T Cells

    Science.gov (United States)

    Perišić Nanut, Milica; Sabotič, Jerica; Jewett, Anahid; Kos, Janko

    2014-01-01

    Cysteine cathepsins are lysosomal peptidases involved at different levels in the processes of the innate and adaptive immune responses. Some, such as cathepsins B, L, and H are expressed constitutively in most immune cells. In cells of innate immunity they play a role in cell adhesion and phagocytosis. Other cysteine cathepsins are expressed more specifically. Cathepsin X promotes dendritic cell maturation, adhesion of macrophages, and migration of T cells. Cathepsin S is implicated in major histocompatibility complex class II antigen presentation, whereas cathepsin C, expressed in cytotoxic T lymphocytes and natural killer (NK) cells, is involved in processing pro-granzymes into proteolytically active forms, which trigger cell death in their target cells. The activity of cysteine cathepsins is controlled by endogenous cystatins, cysteine protease inhibitors. Of these, cystatin F is the only cystatin that is localized in endosomal/lysosomal vesicles. After proteolytic removal of its N-terminal peptide, cystatin F becomes a potent inhibitor of cathepsin C with the potential to regulate pro-granzyme processing and cell cytotoxicity. This review is focused on the role of cysteine cathepsins and their inhibitors in the molecular mechanisms leading to the cytotoxic activity of T lymphocytes and NK cells in order to address new possibilities for regulation of their function in pathological processes. PMID:25520721

  11. Recombiner

    International Nuclear Information System (INIS)

    Saalfrank, H.

    1985-01-01

    Air containing hydrogen can be oxidized by heating in a container called a recombiner, in order to avoid the collection of hydrogen. The container is long and a large number of straight heating bars are arranged in parallel in it and they are flanged to a lid. The heating bars are surrounded by tubes, in order to obtain good heat transfer by a narrow annular gap. (orig.) [de

  12. Cysteine cathepsins B and X promote epithelial-mesenchymal transition of tumor cells.

    Science.gov (United States)

    Mitrović, Ana; Pečar Fonović, Urša; Kos, Janko

    2017-09-01

    Cathepsins B and X are lysosomal cysteine carboxypeptidases suggested as having a redundant role in cancer. They are involved in a number of processes leading to tumor progression but their role in the epithelial-mesenchymal transition (EMT) remains unknown. We have investigated the contribution of both cathepsins B and X in EMT using tumor cell lines differing in their expression of epithelial and mesenchymal markers and cell morphology. Higher levels of both cathepsins are shown to promote EMT and are associated with the mesenchymal-like cell phenotype. Moreover, simultaneous knockdown of the two peptidases triggers a reverse, mesenchymal to epithelial transition. Of the two cathepsins, cathepsin B appears to be the stronger promotor of EMT. Furthermore, we evaluated the involvement of cathepsin B and X in the transforming growth factor-β1 (TGF-β1) signaling pathway, one of the key signaling mechanisms triggering EMT in cancer. In MCF-7 cells the expression of cathepsin B was shown to depend on their activation with TGF-β1 while, for cathepsin X, a TGF-β1 independent mechanism of induction during EMT is indicated. EMT is thus shown to be another mechanism linking cathepsins B and X with tumor progression. With silencing of their expression or inhibition of enzymatic activity, the tumor cells could be reverted to less aggressive epithelial-like phenotype. Copyright © 2017 Elsevier GmbH. All rights reserved.

  13. Anti-proliferative activity of recombinant melittin expressed in ...

    African Journals Online (AJOL)

    kesiena

    2012-02-09

    Feb 9, 2012 ... 44 amino acid residues mediated by dipeptidylpeptidase. IV (Vlasak et al., 1983). It has been reported that the melittin exhibits antimicrobial activity and pro- ... Construction of recombinant expression vector. A pair of complementary oligonucleotides named Mel-1 (5′-GAT. CCG GAA TTG GAG CAG TTC ...

  14. Prdm9 controls activation of mammalian recombination hotspots.

    Science.gov (United States)

    Parvanov, Emil D; Petkov, Petko M; Paigen, Kenneth

    2010-02-12

    Mammalian meiotic recombination, which preferentially occurs at specialized sites called hotspots, ensures the orderly segregation of meiotic chromosomes and creates genetic variation among offspring. A locus on mouse chromosome 17, which controls activation of recombination at multiple distant hotspots, has been mapped within a 181-kilobase interval, three of whose genes can be eliminated as candidates. The remaining gene, Prdm9, codes for a zinc finger containing histone H3K4 trimethylase that is expressed in early meiosis and whose deficiency results in sterility in both sexes. Mus musculus exhibits five alleles of Prdm9; human populations exhibit two predominant alleles and multiple minor alleles. The identification of Prdm9 as a protein regulating mammalian recombination hotspots initiates molecular studies of this important biological control system.

  15. A novel clot lysis assay for recombinant plasminogen activator.

    Science.gov (United States)

    Jamialahmadi, Oveis; Fazeli, Ahmad; Hashemi-Najafabadi, Sameereh; Fazeli, Mohammad Reza

    2015-03-01

    Recombinant plasminogen activator (r-PA, reteplase) is an engineered variant of alteplase. When expressed in E. coli, it appears as inclusion bodies that require refolding to recover its biological activity. An important step following refolding is to determine the activity of refolded protein. Current methods for enzymatic activity of thrombolytic drugs are costly and complex. Here a straightforward and low-cost clot lysis assay was developed. It quantitatively measures the activity of the commercial reteplase and is also capable of screening refolding conditions. As evidence for adequate accuracy and sensitivity of the current assay, r-PA activity measurements are shown to be comparable to those obtained from chromogenic substrate assay.

  16. Molecular cloning, characterization and functional analysis of a novel juvenile-specific cathepsin L of Fasciola gigantica.

    Science.gov (United States)

    Sansri, Veerawat; Changklungmoa, Narin; Chaichanasak, Pannigan; Sobhon, Prasert; Meemon, Krai

    2013-10-01

    Cathepsin L proteases are a major class of endopeptidases expressed at a high level in Fasciola parasites. Several isoforms of cathepsin L were detected and they may perform different functions during the parasite development. In this study, a complete cDNA encoding a cathepsin L protease was cloned from a newly excysted juvenile (NEJ) cDNA library of Fasciola gigantica and named FgCatL1H. It encoded a 326 amino acid preproenzyme which shared 62.8-83.1% and 39.5-42.9% identity to Fasciola spp. and mammalian cathepsins L, respectively. All functionally important residues previously described for cathepsin L were conserved in FgCatL1H. Phylogenetic analysis demonstrated that FgCatL1H belonged to a distinct group, clade 4, with respect to adult and other juvenile Fasciola cathepsin L genes. FgCatL1H expression was detected by RT-PCR, using gene specific primers, in metacercariae and NEJ, and the expression gradually decreased in advanced developmental stages. A recombinant proFgCatL1H (rproFgCatL1H) was expressed in the yeast Pichia pastoris, affinity purified, and found to migrate in SDS-PAGE at approximately 47.6 and 38.3kDa in glycosylated and deglycosylated forms, respectively. The molecular mass of the activated mature rFgCatL1H in glycosylated form was approximately 40.7kDa. Immunoblotting and immunohistochemistry using rabbit antibodies against rproFgCatL1H showed that FgCatL1H was predominantly expressed in epithelial cells of the digestive tract of metacercariae, NEJs and juveniles of F. gigantica. FgCatL1H could cleave the synthetic fluorogenic substrate Z-Phe-Arg-MCA preferentially over Z-Gly-Pro-Arg-MCA at an optimum pH of 6.5. It also showed hydrolytic activity against native substrates, including type I collagen, laminin, and immunoglobulin G (IgG) in vitro, suggesting possible roles in host tissue migration and immune evasion. Therefore, the FgCatL1H is a possible target for vaccine and chemotherapy for controlling F. gigantica infection. Copyright

  17. Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer

    International Nuclear Information System (INIS)

    Andrade, Sheila Siqueira; Gouvea, Iuri Estrada; Silva, Mariana Cristina C.; Castro, Eloísa Dognani; Paula, Cláudia A. A. de; Okamoto, Debora; Oliveira, Lilian; Peres, Giovani Bravin; Ottaiano, Tatiana; Facina, Gil; Nazário, Afonso Celso Pinto; Campos, Antonio Hugo J. F. M.; Paredes-Gamero, Edgar Julian; Juliano, Maria; Silva, Ismael D. C. G. da; Oliva, Maria Luiza V.; Girão, Manoel J. B. C.

    2016-01-01

    Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and −4 are highly expressed, but PAR-3 shows low expression and unclear functions. Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFβ monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and −4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFβ in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells. The online version of this article (doi:10.1186/s12885-016-2203-7) contains supplementary material, which is available to authorized users

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

    International Nuclear Information System (INIS)

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

    1989-01-01

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

  19. Optimization of triazine nitriles as rhodesain inhibitors: structure-activity relationships, bioisosteric imidazopyridine nitriles, and X-ray crystal structure analysis with human cathepsin L.

    Science.gov (United States)

    Ehmke, Veronika; Winkler, Edwin; Banner, David W; Haap, Wolfgang; Schweizer, W Bernd; Rottmann, Matthias; Kaiser, Marcel; Freymond, Céline; Schirmeister, Tanja; Diederich, François

    2013-06-01

    The cysteine protease rhodesain of Trypanosoma brucei parasites causing African sleeping sickness has emerged as a target for the development of new drug candidates. Based on a triazine nitrile moiety as electrophilic headgroup, optimization studies on the substituents for the S1, S2, and S3 pockets of the enzyme were performed using structure-based design and resulted in inhibitors with inhibition constants in the single-digit nanomolar range. Comprehensive structure-activity relationships clarified the binding preferences of the individual pockets of the active site. The S1 pocket tolerates various substituents with a preference for flexible and basic side chains. Variation of the S2 substituent led to high-affinity ligands with inhibition constants down to 2 nM for compounds bearing cyclohexyl substituents. Systematic investigations on the S3 pocket revealed its potential to achieve high activities with aromatic vectors that undergo stacking interactions with the planar peptide backbone forming part of the pocket. X-ray crystal structure analysis with the structurally related enzyme human cathepsin L confirmed the binding mode of the triazine ligand series as proposed by molecular modeling. Sub-micromolar inhibition of the proliferation of cultured parasites was achieved for ligands decorated with the best substituents identified through the optimization cycles. In cell-based assays, the introduction of a basic side chain on the inhibitors resulted in a 35-fold increase in antitrypanosomal activity. Finally, bioisosteric imidazopyridine nitriles were studied in order to prevent off-target effects with unselective nucleophiles by decreasing the inherent electrophilicity of the triazine nitrile headgroup. Using this ligand, the stabilization by intramolecular hydrogen bonding of the thioimidate intermediate, formed upon attack of the catalytic cysteine residue, compensates for the lower reactivity of the headgroup. The imidazopyridine nitrile ligand showed

  20. Localization of nuclear cathepsin L and its association with disease progression and poor outcome in colorectal cancer.

    LENUS (Irish Health Repository)

    Sullivan, Shane

    2012-02-01

    Previous in vitro studies have identified a nuclear isoform of Cathepsin L. The aim of this study was to examine if nuclear Cathepsin L exists in vivo and examine its association with clinical, pathological and patient outcome data. Cellular localization (nuclear and cytoplasmic) and expression levels v of Cathespin L in 186 colorectal cancer cases using immunohistochemistry. The molecular weight and activity of nuclear and cytoplasmic Cathepsin L in vivo and in vitro were assessed by Western blotting and ELISA, respectively. Epithelial nuclear staining percentage (p = 0.04) and intensity (p = 0.006) increased with advancing tumor stage, whereas stromal cytoplasmic staining decreased (p = 0.02). Using multivariate statistical analysis, survival was inversely associated with staining intensity in the epithelial cytoplasm (p = 0.01) and stromal nuclei (p = 0.007). In different colorectal cell lines and in vivo tumors, pro- and active Cathepsin L isoforms were present in both the cytoplasm and nuclear samples, with pro-Cathepsin L at 50 kDa and active Cathepsin L at 25 kDa. Purified nuclear and cytoplasmic fractions from cell lines and tumors showed active Cathepsin L activity. The identification of nuclear Cathepsin L may play an important prognostic role in colorectal disease progression and patient outcome. Moreover, these findings suggest that altering active nuclear Cathepsin L may significantly influence disease progression.

  1. Cathepsin S Is Involved in Th17 Differentiation Through the Upregulation of IL-6 by Activating PAR-2 after Systemic Exposure to Lipopolysaccharide from Porphyromonas gingivalis

    Directory of Open Access Journals (Sweden)

    Masato Dekita

    2017-07-01

    Full Text Available Positive links have been found between periodontitis and numerous diseases in humans via persistent inflammation throughout the body. However, the main factors responsible for maintaining this pro-inflammatory condition are poorly understood. The spleen, the largest secondary immune organ, is a central hub regulating the immune response/inflammation due to the dendritic cell (DC response to CD4+ T cell subtype differentiation, and lysosomal proteinase cathepsin S (CatS is known to be involved in DC functions. In the present study, we found that CatS-induced IL-6 production by splenic DCs subsequently promotes Th17 differentiation, in response to systemic exposure to lipopolysaccharide derived from Porphyromonas gingivalis (PgLPS. The population of CD11c+ DCs was significantly increased in the splenic marginal zone (MZ locally of wild-type (DBA/2 mice with splenomegaly but not in that of CatS deficient (CatS-/- mice after systemic exposure to PgLPS for 7 consecutive days (5 mg/kg/day, intraperitoneal. Similarly, the population of Th17+CD4+ T cells was also significantly increased in the splenic MZ of wild-type mice but not in that of CatS-/- mice after PgLPS exposure. Furthermore, the increase in the Th17+ CD4+ T cell population paralleled increases in the levels of CatS and IL-6 in CD11c+ cells in the splenic MZ. In isolated primary splenic CD11c+ cells, the mRNA expression and the production of IL-6 was dramatically increased in wild-type mice but not in CatS-/- mice after direct stimulation with PgLPS (1 μg/ml, and this PgLPS-induced increase in the IL-6 expression was completely abolished by pre-treatment with Z-Phe-Leu-COCHO (Z-FL, the specific inhibitor of CatS. The PgLPS activated protease-activated receptor (PAR 2 in the isolated splenic CD11c+ cells was also significantly inhibited by CatS deficiently. In addition, the PgLPS-induced increase in the IL-6 production by splenic CD11c+ cells was completely abolished by pre-treatment with

  2. Molecular cloning and functional characterization of cathepsin D from sea cucumber Apostichopus japonicus.

    Science.gov (United States)

    Yu, Cuiping; Cha, Yue; Wu, Fan; Xu, Xianbing; Qin, Lei; Du, Ming

    2017-11-01

    Cathepsin D (CTSD, EC 3.4.23.5) belongs to aspartic protease family, which is located in lysosomes and is distributed in diverse tissues and cells. CTSD has a wide variety of physiological functions, owing to its proteolytic activity in degradating proteins and peptides. In the current study, the full length cDNA of sea cucumber (Apostichopus japonicus) cathepsin D (AjCTSD) was firstly cloned, then the association between AjCTSD and sea cucumber autolysis was investigated. The full length cDNA of AjCTSD was 2896 bp, with an open reading frame (ORF) for 391 amino acids. AjCTSD was widely expressed in body wall, muscle and intestine; the expression level was the highest in intestine, followed by muscle and body wall. Compared to fresh tissues, AjCTSD expression levels were significantly increased in all examined autolytic tissues. The purified recombinant AjCTSD promoted the degradation of sea cucumber muscle. In conclusion, AjCTSD contributed to sea cucumber muscle autolysis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. Heparin modulates the endopeptidase activity of Leishmania mexicana cysteine protease cathepsin L-Like rCPB2.8.

    Directory of Open Access Journals (Sweden)

    Wagner A S Judice

    Full Text Available Cysteine protease B is considered crucial for the survival and infectivity of the Leishmania in its human host. Several microorganism pathogens bind to the heparin-like glycosaminoglycans chains of proteoglycans at host-cell surface to promote their attachment and internalization. Here, we have investigated the influence of heparin upon Leishmania mexicana cysteine protease rCPB2.8 activity.THE DATA ANALYSIS REVEALED THAT THE PRESENCE OF HEPARIN AFFECTS ALL STEPS OF THE ENZYME REACTION: (i it decreases 3.5-fold the k 1 and 4.0-fold the k -1, (ii it affects the acyl-enzyme accumulation with pronounced decrease in k 2 (2.7-fold, and also decrease in k 3 (3.5-fold. The large values of ΔG  =  12 kJ/mol for the association and dissociation steps indicate substantial structural strains linked to the formation/dissociation of the ES complex in the presence of heparin, which underscore a conformational change that prevents the diffusion of substrate in the rCPB2.8 active site. Binding to heparin also significantly decreases the α-helix content of the rCPB2.8 and perturbs the intrinsic fluorescence emission of the enzyme. The data strongly suggest that heparin is altering the ionization of catalytic (Cys(25-S(-/(His(163-Im(+ H ion pair of the rCPB2.8. Moreover, the interaction of heparin with the N-terminal pro-region of rCPB2.8 significantly decreased its inhibitory activity against the mature enzyme.Taken together, depending on their concentration, heparin-like glycosaminoglycans can either stimulate or antagonize the activity of cysteine protease B enzymes during parasite infection, suggesting that this glycoconjugate can anchor parasite cysteine protease at host cell surface.

  4. Cathepsin L of Triatoma brasiliensis (Reduviidae, Triatominae): sequence characterization, expression pattern and zymography.

    Science.gov (United States)

    Waniek, Peter J; Pacheco Costa, Juliana E; Jansen, Ana M; Costa, Jane; Araújo, Catarina A C

    2012-01-01

    Triatoma brasiliensis is considered one of the main vectors of Chagas disease commonly found in semi-arid areas of northeastern Brazil. These insects use proteases, such as carboxypeptidase B, aminopeptidases and different cathepsins for blood digestion. In the present study, two genes encoding cathepsin L from the midgut of T. brasiliensis were identified and characterized. Mature T. brasiliensis cathepsin L-like proteinases (TBCATL-1, TBCATL-2) showed a high level of identity to the cathepsin L-like proteinases of other insects, with highest similarity to Rhodnius prolixus. Both cathepsin L transcripts were highly abundant in the posterior midgut region, the main region of the blood digestion. Determination of the pH in the whole intestine of unfed T. brasiliensis revealed alkaline conditions in the anterior midgut region (stomach) and acidic conditions in the posterior midgut region (small intestine). Gelatine in-gel zymography showed the activity of at least four distinct proteinases in the small intestine and the cysteine proteinase inhibitors transepoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64) and cathepsin B inhibitor and N-(l-3-trans-propylcarbamoyl-oxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074) were employed to characterize enzymatic activity. E-64 fully inhibited cysteine proteinase activity, whereas in the samples treated with CA-074 residual proteinase activity was detectable. Thus, proteolytic activity could at least partially be ascribed to cathepsin L. Western blot analysis using specific anti cathepsin L antibodies confirmed the presence of cathepsin L in the lumen of the small intestine of the insects. Copyright © 2011 Elsevier Ltd. All rights reserved.

  5. Recombination activating activity of XRCC1 analogous genes in X-ray sensitive and resistant CHO cell lines

    International Nuclear Information System (INIS)

    Golubnitchaya-Labudova, O.; Hoefer, M.; Portele, A.; Vacata, V.; Rink, H.; Lubec, G.

    1997-01-01

    The XRCC1 gene (X-ray repair cross complementing) complements the DNA repair deficiency of the radiation sensitive Chinese hamster ovary (CHO) mutant cell line EM9 but the mechanism of the correction is not elucidated yet. XRCC1 shows substantial homology to the RAG2 gene (recombination activating gene) and we therefore tried to answer the question, whether structural similarities (sequence of a putative recombination activating domain, aa 332-362 for XRCC1 and aa 286-316 in RAG2) would reflect similar functions of the homologous, putative recombination activating domain. PCR experiments revealed that no sequence homologous to the structural part of human XRCC1 was present in cDNA of CHO. Differential display demonstrated two putative recombination activating in the parental CHO line AA8 and one in the radiosensitive mutant EM9. Southern blot experiments showed the presence of several genes with partial homology to human XRCC1. Recombination studies consisted of expressing amplified target domains within chimeric proteins in recA - bacteria and subsequent detection of recombination events by sequencing the recombinant plasmids. Recombination experiments demonstrated recombination activating activity of all putative recombination activating domains amplified from AA8 and EM9 genomes as reflected by deletions within the inserts of the recombinant plasmids. The recombination activating activity of XRCC1 analogues could explain a mechanism responsible for the correction of the DNA repair defect in EM9. (author)

  6. Mechanism of action of recombinant activated factor VII: an update.

    Science.gov (United States)

    Hedner, Ulla

    2006-01-01

    Bleeding episodes in patients with hemophilia and inhibitors must be managed using agents that are hemostatically active in the absence of factor VIII or IX. Activated prothrombin complex concentrates have long been used in this context. However, the search for safer and more effective agents has led to the development of recombinant activated factor VII (rFVIIa; NovoSeven, Novo Nordisk, Bagsvaerd, Denmark). This paper presents an update on the mechanism of action of rFVIIa, and describes how pharmacologic doses of this agent enhance thrombin production and thus contribute to the development of a stable, lysis-resistant fibrin plug at the site of vessel damage. This mechanism explains the reported efficacy of rFVIIa in a range of clinical situations characterized by impaired thrombin generation.

  7. Low-Cost Method to Monitor Patient Adherence to HIV Antiretroviral Therapy Using Multiplex Cathepsin Zymography.

    Science.gov (United States)

    Platt, Manu O; Evans, Denise; Keegan, Philip M; McNamara, Lynne; Parker, Ivana K; Roberts, LaDeidra M; Caulk, Alexander W; Gleason, Rudolph L; Seifu, Daniel; Amogne, Wondwossen; Penny, Clement

    2016-01-01

    Monitoring patient adherence to HIV antiretroviral therapy (ART) by patient survey is inherently error prone, justifying a need for objective, biological measures affordable in low-resource settings where HIV/AIDS epidemic is highest. In preliminary studies conducted in Ethiopia and South Africa, we observed loss of cysteine cathepsin activity in peripheral blood mononuclear cells of HIV-positive patients on ART. We optimized a rapid protocol for multiplex cathepsin zymography to quantify cysteine cathepsins, and prospectively enrolled 350 HIV-positive, ART-naïve adults attending the Themba Lethu Clinic, Johannesburg, South Africa, to test if suppressed cathepsin activity could be a biomarker of ART adherence (103 patients were included in final analysis). Poor adherence was defined as detectable viral load (>400 copies/ml) or simplified medication adherence questionnaire, 4-6 months after ART initiation. 86 % of patients with undetectable viral loads after 6 months were cathepsin negative, and cathepsin-positive patients were twice as likely to have detectable viral loads (RR 2.32 95 % CI 1.26-4.29). Together, this demonstrates proof of concept that multiplex cathepsin zymography may be an inexpensive, objective method to monitor patient adherence to ART. Low cost of this electrophoresis-based assay makes it a prime candidate for implementation in resource-limited settings.

  8. Low cost method to monitor patient adherence to HIV antiretroviral therapy using multiplex cathepsin zymography

    Science.gov (United States)

    Platt, Manu O.; Evans, Denise; Keegan, Philip M.; McNamara, Lynne; Parker, Ivana K.; Roberts, LaDeidra M.; Caulk, Alexander W.; Gleason, Rudolph L.; Seifu, Daniel; Amogne, Wondwossen; Penny, Clement

    2015-01-01

    Monitoring patient adherence to HIV antiretroviral therapy (ART) by patient survey is inherently error-prone, justifying a need for objective, biological measures affordable in low resource settings where HIV/AIDS epidemic is highest. In preliminary studies conducted in Ethiopia and South Africa, we observed loss of cysteine cathepsin activity in peripheral blood mononuclear cells (PBMCs) of HIV-positive patients on ART. We optimized a rapid protocol for multiplex cathepsin zymography to quantify cysteine cathepsins, and prospectively enrolled 350 HIV-positive, ART naïve adults attending the Themba Lethu Clinic, Johannesburg, South Africa, to test if suppressed cathepsin activity could be a biomarker of ART adherence (103 patients were included in final analysis). Poor adherence was defined as detectable viral load (>400 copies/ml) or simplified medication adherence questionnaire (SMAQ), 4–6 months after ART initiation. 86% of patients with undetectable viral loads after 6 months were cathepsin negative, and cathepsin positive patients were twice as likely to have detectable viral loads (RR 2.32 95% CI 1.26–4.29). Together, this demonstrates proof of concept that multiplex cathepsin zymography may be an inexpensive, objective method to monitor patient adherence to ART. Low cost of this electrophoresis based assay makes it a prime candidate for implementation in resource limited settings. PMID:26589706

  9. Production of biologically active recombinant human factor H in Physcomitrella.

    Science.gov (United States)

    Büttner-Mainik, Annette; Parsons, Juliana; Jérôme, Hanna; Hartmann, Andrea; Lamer, Stephanie; Schaaf, Andreas; Schlosser, Andreas; Zipfel, Peter F; Reski, Ralf; Decker, Eva L

    2011-04-01

    The human complement regulatory serum protein factor H (FH) is a promising future biopharmaceutical. Defects in the gene encoding FH are associated with human diseases like severe kidney and retinal disorders in the form of atypical haemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis II (MPGN II) or age-related macular degeneration (AMD). There is a current need to apply intact full-length FH for the therapy of patients with congenital or acquired defects of this protein. Application of purified or recombinant FH (rFH) to these patients is an important and promising approach for the treatment of these diseases. However, neither protein purified from plasma of healthy individuals nor recombinant protein is currently available on the market. Here, we report the first stable expression of the full-length human FH cDNA and the subsequent production of this glycoprotein in a plant system. The moss Physcomitrella patens perfectly suits the requirements for the production of complex biopharmaceuticals as this eukaryotic system not only offers an outstanding genetical accessibility, but moreover, proteins can be produced safely in scalable photobioreactors without the need for animal-derived medium compounds. Transgenic moss lines were created, which express the human FH cDNA and target the recombinant protein to the culture supernatant via a moss-derived secretion signal. Correct processing of the signal peptide and integrity of the moss-produced rFH were verified via peptide mapping by mass spectrometry. Ultimately, we show that the rFH displays complement regulatory activity comparable to FH purified from plasma. © 2010 The Authors. Plant Biotechnology Journal © 2010 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

  10. Imaging Primary Mouse Sarcomas After Radiation Therapy Using Cathepsin-Activatable Fluorescent Imaging Agents

    Energy Technology Data Exchange (ETDEWEB)

    Cuneo, Kyle C. [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Mito, Jeffrey K.; Javid, Melodi P. [Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States); Ferrer, Jorge M. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Kim, Yongbaek [Department of Clinical Pathology, College of Veterinary Medicine, Seoul National University, Seoul (Korea, Republic of); Lee, W. David [The David H. Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Bawendi, Moungi G. [Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts (United States); Brigman, Brian E. [Department of Orthopedic Surgery, Duke University School of Medicine, Durham, North Carolina (United States); Kirsch, David G., E-mail: david.kirsch@duke.edu [Department of Radiation Oncology, Duke University School of Medicine, Durham, North Carolina (United States); Department of Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina (United States)

    2013-05-01

    Purpose: Cathepsin-activated fluorescent probes can detect tumors in mice and in canine patients. We previously showed that these probes can detect microscopic residual sarcoma in the tumor bed of mice during gross total resection. Many patients with soft tissue sarcoma (STS) and other tumors undergo radiation therapy (RT) before surgery. This study assesses the effect of RT on the ability of cathepsin-activated probes to differentiate between normal and cancerous tissue. Methods and Materials: A genetically engineered mouse model of STS was used to generate primary hind limb sarcomas that were treated with hypofractionated RT. Mice were injected intravenously with cathepsin-activated fluorescent probes, and various tissues, including the tumor, were imaged using a hand-held imaging device. Resected tumor and normal muscle samples were harvested to assess cathepsin expression by Western blot. Uptake of activated probe was analyzed by flow cytometry and confocal microscopy. Parallel in vitro studies using mouse sarcoma cells were performed. Results: RT of primary STS in mice and mouse sarcoma cell lines caused no change in probe activation or cathepsin protease expression. Increasing radiation dose resulted in an upward trend in probe activation. Flow cytometry and immunofluorescence showed that a substantial proportion of probe-labeled cells were CD11b-positive tumor-associated immune cells. Conclusions: In this primary murine model of STS, RT did not affect the ability of cathepsin-activated probes to differentiate between tumor and normal muscle. Cathepsin-activated probes labeled tumor cells and tumor-associated macrophages. Our results suggest that it would be feasible to include patients who have received preoperative RT in clinical studies evaluating cathepsin-activated imaging probes.

  11. Purification and Characterization of Cathepsin B from the Muscle of Horse Mackerel Trachurus japonicus

    Directory of Open Access Journals (Sweden)

    Asami Yoshida

    2015-10-01

    Full Text Available An endogenous protease in fish muscle, cathepsin B, was partially purified and characterized from horse mackerel meat. On SDS-PAGE of the purified enzyme under reducing conditions, main protein bands were detected at 28 and 6 kDa and their respective N-terminal sequences showed high homology to heavy and light chains of cathepsin B from other species. This suggested that horse mackerel cathepsin B formed two-chain forms, similar to mammalian cathepsin Bs. Optimum pH and temperature of the enzyme were 5.0 and 50 °C, respectively. A partial cDNA encoding the amino acid sequence of 215 residues for horse mackerel cathepsin B was obtained by RT-PCR and cloned. The deduced amino acid sequence contains a part of light and heavy chains of cathepsin B. The active sites and an N-glycosylation site were conserved across species. We also confirmed that the modori phenomenon was avoided by CA-074, a specific inhibitor for cathepsin B. Therefore, our results suggest that natural cysteine protease inhibitor(s, such as oryzacystatin derived from rice, can apply to thermal-gel processing of horse mackerel to avoid the modori phenomenon. Meanwhile, this endogenous protease may be used for food processing, such as weaning meal and food for the elderly.

  12. Cathepsin L is an immune-related protein in Pacific abalone (Haliotis discus hannai)--Purification and characterization.

    Science.gov (United States)

    Shen, Jian-Dong; Cai, Qiu-Feng; Yan, Long-Jie; Du, Cui-Hong; Liu, Guang-Ming; Su, Wen-Jin; Ke, Caihuan; Cao, Min-Jie

    2015-12-01

    Cathepsin L, an immune-related protein, was purified from the hepatopancreas of Pacific abalone (Haliotis discus hannai) by ammonium sulfate precipitation and column chromatographies of SP-Sepharose and Sephacryl S-200 HR. Purified cathepsin L appeared as two bands with molecular masses of 28.0 and 28.5 kDa (namely cathepsin La and Lb) on SDS-PAGE under reducing conditions, suggesting that it is a glycoprotein. Peptide mass fingerprinting (PMF) analysis revealed that peptide fragments of 95 amino acid residues was high similarity to cathepsin L of pearl oyster (Pinctada fucata). The optimal temperature and pH of cathepsin L were 35 °C and pH 5.5. Cathepsin L was particularly inhibited by cysteine proteinase inhibitors of E-64 and leupeptin, while it was activated by metalloproteinase inhibitors EDTA and EGTA. The full-length cathepsin L cDNA was further cloned from the hepatopancreas by rapid PCR amplification of cDNA ends (RACE). The open reading frame of the enzyme was 981 bp, encoding 327 amino acid residues, with a conserved catalytic triad (Cys134, His273 and Asn293), a potential N-glycosylation site and conserved ERFNIN, GNYD, and GCGG motifs, which are characteristics of cathepsin L. Western blot and proteinase activity analysis revealed that the expression and enzyme activity of cathepsin L were significantly up-regulated in hepatopancreas at 8 h following Vibrio parahaemolyticus infection, demonstrating that cathepsin L is involved in the innate immune system of abalone. Our present study for the first time reported the purification, characterization, molecular cloning, and tissue expression of cathepsin L in abalone. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. N-terminally truncated forms of human cathepsin F accumulate in aggresome-like inclusions.

    Science.gov (United States)

    Jerič, Barbara; Dolenc, Iztok; Mihelič, Marko; Klarić, Martina; Zavašnik-Bergant, Tina; Gunčar, Gregor; Turk, Boris; Turk, Vito; Stoka, Veronika

    2013-10-01

    The contribution of individual cysteine cathepsins as positive mediators of programmed cell death is dependent on several factors, such as the type of stimuli, intensity and duration of the stimulus, and cell type involved. Of the eleven human cysteine cathepsins, cathepsin F is the only cathepsin that exhibits an extended N-terminal proregion, which contains a cystatin-like domain. We predicted that the wild-type human cathepsin F contains three natively disordered regions within the enzyme's propeptide and various amino acid stretches with high fibrillation propensity. Wild-type human cathepsin F and its N-terminally truncated forms, Ala(20)-Asp(484) (Δ(19)CatF), Pro(126)-Asp(484) (Δ(125)CatF), and Met(147)-Asp(484) (Δ(146)CatF) were cloned into the pcDNA3 vector and overexpressed in HEK 293T cells. Wild-type human cathepsin F displayed a clear vesicular labeling and colocalized with the LAMP2 protein, a lysosomal marker. However, all three N-terminally truncated forms of human cathepsin F were recovered as insoluble proteins, suggesting that the deletion of at least the signal peptides (Δ(19)CatF), results in protein aggregation. Noteworthy, they concentrated large perinuclear-juxtanuclear aggregates that accumulated within aggresome-like inclusions. These inclusions showed p62-positive immunoreactivity and were colocalized with the autophagy marker LC3B, but not with the LAMP2 protein. In addition, an approximately 2-3 fold increase in DEVDase activity was not sufficient to induce apoptotic cell death. These results suggested the clearance of the N-terminally truncated forms of human cathepsin F via the autophagy pathway, underlying its protective and prosurvival mechanisms. Copyright © 2013 The Authors. Published by Elsevier B.V. All rights reserved.

  14. Fasciola gigantica cathepsin B5 is an acidic endo- and exopeptidase of the immature and mature parasite.

    Science.gov (United States)

    Siricoon, Sinee; Vichasri Grams, Suksiri; Lertwongvisarn, Kittisak; Abdullohfakeeyah, Muntana; Smooker, Peter M; Grams, Rudi

    2015-12-01

    Cysteine proteases of the liver fluke Fasciola have been described as essential molecules in the infection process of the mammalian host. Destinct cathepsin Bs, which are already expressed in the metacercarial stage and released by the newly excysted juvenile are major actors in this process. Following infection their expression is stopped and the proteins will not be detectable any longer after the first month of development. On the contrary, the novel cathepsin B5 of Fasciola gigantica (FgCB5) described in this work was also found expressed in later juvenile stages and the mature worm. Like all previously described Fasciola family members it was located in the cecal epithelium of the parasite. Western blot analysis of adult antigen preparations detected procathepsin B5 in crude worm extract and in small amounts in the ES product. In support of these data, the sera of infected rabbits and mice were reactive with recombinant FgCB5 in Western blot and ELISA. Biochemical analysis of yeast-expressed FgCB5 revealed that it has properties of a lysosomal hydrolase optimized for activity at acid pH and that it is able to efficiently digest a broad spectrum of host proteins. Unlike previously characterized Fasciola family members FgCB5 carries a histidine doublet in the occluding loop equivalent to residues His110 and His111 of human mature cathepsin B and consequently showed substantial carboxydipeptidyl activity which depends on these two residues. Copyright © 2015 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

  15. Recombinant activated factor VII: 30 years of research and innovation.

    Science.gov (United States)

    Hedner, Ulla

    2015-06-01

    Recombinant activated factor VII (rFVIIa) was initially developed to treat bleeding episodes in patients with congenital haemophilia and inhibitors. The story of its development began in the 1970s, when FVIIa was identified as one of the activated coagulation factors that has minimal potential for inducing thromboembolic side-effects. Extensive research over the last 30 years has greatly increased our knowledge of the characteristics of FVII, its activation, and the mechanisms by which rFVIIa restores haemostasis. In haemophilia, the haemostatic effect of rFVIIa is mediated via binding to thrombin-activated platelets at the site of injury, thereby enhancing thrombin generation also in the absence of factor (F) VIII or FIX. The mechanism of action of rFVIIa has also allowed its successful use in other clinical scenarios characterised by impaired thrombin generation, and its licensed uses have now been extended to acquired haemophilia, congenital FVII deficiency and Glanzmann's thrombasthenia. Copyright © 2015 Elsevier Ltd. All rights reserved.

  16. Permanent Genetic Access to Transiently Active Neurons via TRAP: Targeted Recombination in Active Populations

    OpenAIRE

    Guenthner, Casey J.; Miyamichi, Kazunari; Yang, Helen H.; Heller, H. Craig; Luo, Liqun

    2013-01-01

    Targeting genetically encoded tools for neural circuit dissection to relevant cellular populations is a major challenge in neurobiology. We developed a new approach, Targeted Recombination in Active Populations (TRAP), to obtain genetic access to neurons that were activated by defined stimuli. This method utilizes mice in which the tamoxifen-dependent recombinase CreERT2 is expressed in an activity-dependent manner from the loci of the immediate early genes Arc and Fos. Active cells that expr...

  17. Gelatin Zymography Using Leupeptin for the Detection of Various Cathepsin L Forms.

    Science.gov (United States)

    Hashimoto, Yoko

    2017-01-01

    Zymography is a highly sensitive method to assess the activities as well as molecular weights of enzymes in crude biological fluids and tissue extracts. Cathepsin L is a lysosomal cysteine proteinase that is optimally active at slightly acidic pH and is highly unstable in alkaline solutions such as electrode buffer (pH 8.3). Large amounts of cathepsin L are secreted by various cancer cells, where it promotes invasion and metastasis. Leupeptin is a tight-binding inhibitor of cysteine proteinases, and its complex with cathepsin L is stable in alkaline solutions. Moreover, leupeptin can be easily removed from the complex because it is a reversibly binding inhibitor. In addition, leupeptin is too small to influence the electrode migration distance of the complex with cathepsin L on a sodium dodecyl sulfate-polyacrylamide gel. Here, a novel gelatin zymography technique that employs leupeptin to detect pro-, intermediate, and mature cathepsin L forms on the basis of their gelatinolytic activities is described. Further, the differences in the glycosylation, phosphorylation, and processing statuses of lysosomal and secreted cathepsin L forms isolated from cultured HT 1080 cells are demonstrated using this method.

  18. Purification and characterization of cathepsin B from the skeletal muscles of agama stellio stellio

    International Nuclear Information System (INIS)

    El-Jassabi, S.; Abu-Ghalyun, Y.

    1997-01-01

    1. Cathepsin b from the muscles of Jordanian lizard Agama stellio stellio was purified to homogeneity by a series of column chromatography on DEAE-sephadex, thio propyl sepharose and sephadex G-100 2. The molecular weight of cathepsin B isolated was to be 31800 dalton by using SDS-PAGE, and 33000 dalton by gel filtration, and its isoelectric point was measured to be 4.2 by isoelectric focusing. 3. Cathepsin B had ph optimum of 5.5, required a thiol-reducing reagent for activation and was inhibited by thiol-protease inhibitors. 4. The Km and K cat values for Z-Phe-Arg-Mca were determined to be 0.161mM and 238 S -1 . 5. Cathepsin B acted on oligopeptide substrates mainly as di peptidyl carboxypeptidase. (authors). 22 refs., 3 tabs., 2 figs

  19. Evidence supporting the use of recombinant activated factor VII in congenital bleeding disorders

    DEFF Research Database (Denmark)

    Johansson, Pär I; Ostrowski, Sisse R

    2010-01-01

    Recombinant activated factor VII (rFVIIa, NovoSeven) was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX.......Recombinant activated factor VII (rFVIIa, NovoSeven) was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX....

  20. Recombinant activated factor VII in cardiac surgery: single-center experience.

    Science.gov (United States)

    Singh, Sarvesh Pal; Chauhan, Sandeep; Choudhury, Minati; Malik, Vishwas; Choudhary, Shiv Kumar

    2014-02-01

    The widespread off-label use of recombinant activated factor VII for the control of refractory postoperative hemorrhage continues despite a warning from the Food and Drug Administration. Although effective in reducing the need for transfusion of blood and blood products, safety concerns still prevail. To compare the dosing and efficacy of recombinant activated factor VII between pediatric and adult patients, and in the operating room and intensive care unit. The records of 69 patients (33 children and 36 adults) who underwent cardiovascular surgery and received recombinant activated factor VII were reviewed retrospectively. The dose of recombinant activated factor VII, mediastinal drainage, use of blood and blood products, incidence of thrombosis, and 28-day mortality were studied. the efficacy of recombinant activated factor VII was comparable in adults and children, despite the lower dose in adults. Prophylactic use of recombinant activated factor VII decreased the incidence of mediastinal exploration and the duration of intensive care unit stay. A 4.3% incidence of thrombotic complications was observed in this study. The efficacious dose of recombinant activated factor VII is much less in adults compared to children. Prophylactic use of recombinant activated factor VII decreases the dose required, the incidence of mediastinal exploration, and intensive care unit stay, with no survival benefit.

  1. Studying of the standardization principles of pharmacological activity of recombinant erythropoietin preparations

    OpenAIRE

    A. K. Yakovlev; L. A. Gayderova; N. A. Alpatova; T. N. Lobanova; E. L. Postnova; E. I. Yurchikova; T. A. Batuashvili; R. A. Volkova; V. N. Podkuiko; Yu. V. Olefir

    2016-01-01

    Analysis of the publications devoted to the structure, functions, mechanism of action of erythropoietin is given in the article. Erythropoietin preparations derived from recombinant DNA technology are a mixture of isoforms with different biological activity, which determine the biological properties pharmacological activity, pharmacokinetics, efficacy and safety of medicinal product. Erythropoietin preparations derived by using recombinant DNA technology are a mixture of isoforms with differe...

  2. [Research progress on cathepsin F of parasitic helminths].

    Science.gov (United States)

    Qu, Zi-Gang; Fu, Bao-Quan

    2013-10-01

    Cathepsin F is an important member of papain-like subfamily in cysteine protease family. Cathepsin F of helminth parasites can hydrolyze the specific substrate, degrade host protein such as hemoglobin for nutrition, and be involved in invasion into host tissue. Therefore, cathepsin F serves as a potential target for parasitic disease immunodiagnosis, vaccine design and anti-parasite drug screening. This article reviews the structural characteristics and mechanisms of cathepsin F, and research advances on cathepsin F of parasitic helminths.

  3. Biochemical characterization and structural modeling of human cathepsin E variant 2 in comparison to the wild-type protein

    Science.gov (United States)

    Puizdar, Vida; Zajc, Tajana; Žerovnik, Eva; Renko, Miha; Pieper, Ursula; Eswar, Narayanan; Šali, Andrej; Dolenc, Iztok; Turk, Vito

    2014-01-01

    Cathepsin E splice variant 2 appears in a number of gastric carcinoma. Here, we report detecting this variant in HeLa cells using polyclonal antibodies and biotinylated inhibitor pepstatin A. An overexpression of GFP fusion proteins of cathepsin E and its splice variant within HEK-293T cells was performed to show their localization. Their distribution under a fluorescence microscope showed that they are colocalized. We also expressed variant 1 and variant 2 of cathepsins E, with propeptide and without it, in Echerichia coli. After refolding from the inclusion bodies, the enzymatic activity and circular dichroism spectra of the splice variant 2 were compared to those of the wild-type mature active cathepsins E. While full-length cathepsin E variant1 is activated at acid pH, the splice variant remains inactive. In contrast to the active cathepsin E, the splice variant 2 predominantly assumes β-sheet structure, prone to oligomerization, at least under in vitro conditions, as shown by Atomic Force Microscopy as shallow disk-like particles. A comparative structure model of splice variant 2 was computed based on its alignment to the known structure of cathepsin E intermediate (Protein Data Bank code 1TZS), and used to rationalize its conformational properties and loss of activity. PMID:22718633

  4. Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5

    Directory of Open Access Journals (Sweden)

    Dewi Fitriani

    2017-09-01

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

  5. Cathepsin D non-proteolytically induces proliferation and migration in human omental microvascular endothelial cells via activation of the ERK1/2 and PI3K/AKT pathways.

    Science.gov (United States)

    Pranjol, Md Zahidul I; Gutowski, Nicholas J; Hannemann, Michael; Whatmore, Jacqueline L

    2018-01-01

    Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. (111)Indium Labelling of Recombinant Activated Coagulation Factor VII

    DEFF Research Database (Denmark)

    Nalla, Amarnadh; Buch, Inge; Sigvardt, Maibritt

    2012-01-01

    The aim of this study is to investigate whether (111)Indium-labelled recombinant FVIIa (rFVIIa) could be a potential radiopharmaceutical for localization of bleeding sources. DTPA-conjugated rFVIIa was radiolabelled with (111)In chloride. In vitro binding efficiency of (111)In-DTPA-rFVIIa to F1A2...

  7. New insights into the evolutionary origins of the recombination-activating gene proteins and V(D)J recombination.

    Science.gov (United States)

    Carmona, Lina Marcela; Schatz, David G

    2017-06-01

    The adaptive immune system of jawed vertebrates relies on V(D)J recombination as one of the main processes to generate the diverse array of receptors necessary for the recognition of a wide range of pathogens. The DNA cleavage reaction necessary for the assembly of the antigen receptor genes from an array of potential gene segments is mediated by the recombination-activating gene proteins RAG1 and RAG2. The RAG proteins have been proposed to originate from a transposable element (TE) as they share mechanistic and structural similarities with several families of transposases and are themselves capable of mediating transposition. A number of RAG-like proteins and TEs with sequence similarity to RAG1 and RAG2 have been identified, but only recently has their function begun to be characterized, revealing mechanistic links to the vertebrate RAGs. Of particular significance is the discovery of ProtoRAG, a transposon superfamily found in the genome of the basal chordate amphioxus. ProtoRAG has many of the sequence and mechanistic features predicted for the ancestral RAG transposon and is likely to be an evolutionary relative of RAG1 and RAG2. In addition, early observations suggesting that RAG1 is able to mediate V(D)J recombination in the absence of RAG2 have been confirmed, implying independent evolutionary origins for the two RAG genes. Here, recent progress in identifying and characterizing RAG-like proteins and the TEs that encode them is summarized and a refined model for the evolution of V(D)J recombination and the RAG proteins is presented. © 2016 Federation of European Biochemical Societies.

  8. Expression and Localization of Cathepsins B, D, and G in Two Cancer Stem Cell Subpopulations in Moderately Differentiated Oral Tongue Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Therese Featherston

    2017-07-01

    Full Text Available AimWe have previously demonstrated the putative presence of two cancer stem cell (CSC subpopulations within moderately differentiated oral tongue squamous cell carcinoma (MDOTSCC, which express components of the renin–angiotensin system (RAS. In this study, we investigated the expression and localization of cathepsins B, D, and G in relation to these CSC subpopulations within MDOTSCC.Methods3,3-Diaminobenzidine (DAB and immunofluorescent (IF immunohistochemical (IHC staining was performed on MDOTSCC samples to determine the expression and localization of cathepsins B, D, and G in relation to the CSC subpopulations. NanoString mRNA analysis and colorimetric in situ hybridization (CISH were used to study their transcripts expression. Enzyme activity assays were performed to determine the activity of these cathepsins in MDOTSCC.ResultsIHC staining demonstrated expression of cathepsins B, D, and G in MDOTSCC. Cathepsins B and D were localized to CSCs within the tumor nests, while cathepsin B was localized to the CSCs within the peri-tumoral stroma, and cathepsin G was localized to the tryptase+ phenotypic mast cells within the peri-tumoral stroma. NanoString and CISH mRNA analyses confirmed transcription activation of cathepsins B, D, and G. Enzyme activity assays confirmed active cathepsins B and D, but not cathepsin G.ConclusionThe presence of cathepsins B and D on the CSCs and cathspsin G on the phenotypic mast cells suggest the presence of bypass loops for the RAS which may be a potential novel therapeutic target for MDOTSCC.

  9. Chemical constituents of the stem bark of Vochysia thyrsoidea Pohl. (Vochysiaceae) and evaluation of their cytotoxicity and inhibitory activity against cathepsins B and K; Constituintes quimicos das cascas do caule de Vochysia thyrsoidea Pohl. (Vochysiaceae) e avaliacao das atividades citotoxica e inibitoria frente as catepsinas B e K

    Energy Technology Data Exchange (ETDEWEB)

    Sousa, Lorena Ramos Freitas de; Silva, Jame' s A. da; Vieira, Paulo Cezar [Universidade Federal de Sao Carlos (UFSCar), SP (Brazil). Departamento de Quimica; Costa, Maisa Borges; Santos, Mirley Luciene dos; Menezes, Antonio Carlos Severo, E-mail: amenezes@ueg.br [Universidade Estadual de Goias (UEG), Anapolis, GO (Brazil). Unidade Universitaria de Ciencias Exatas e Tecnologicas; Sbardelotto, Aline Borba; Pessoa, Claudia do O; Moraes, Manoel Odorico de [Universidade Federal do Ceara (UFC), Fortaleza, CE (Brazil). Fac. de Medicina. Dept. de Fisiologia e Farmacologia

    2014-04-15

    A new flavonoid, catechin-3-O-(3{sup -}O-trans-cinnamoyl)-α-rhamnopyranoside, along with known compounds, catechin-3-O-α-rhamnopyranoside, 3-oxo-urs-12-en-28-oic acid, 2,4,6-trimethoxybenzoic acid, 2-butyl-D-fructofuranoside and 1-butyl-D-fructofuranoside, has been isolated from the stem bark of V. thyrsoidea. These compounds were assayed for inhibition of protease activity (cathepsins B and K) and against cancer cell lines. Catechin-3-O-(3{sup -}O-trans-cinnamoyl)-α-rhamnopyranoside showed moderate inhibitory activity (IC{sub 50} = 62.02 µM) against cathepsin B while 2-butyl-D-fructofuranoside was the most potent against a strain of CNS (SF-295) and human leukemia (HL-60) with IC{sub 50} = 36.80 μM and IC{sub 50} = 25.37 μM, respectively (author)

  10. 78 FR 27977 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Actions Under the NIH...

    Science.gov (United States)

    2013-05-13

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology... Recombinant or Synthetic Nucleic Acid Molecules (NIH Guidelines) SUMMARY: The NIH Office of Biotechnology... of Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, Bethesda...

  11. 75 FR 31795 - Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting

    Science.gov (United States)

    2010-06-04

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology Activities; Recombinant DNA Research: Amended Notice of Meeting ACTION: Notice of cancellation of... information. Dated: May 26, 2010. Jacqueline Corrigan-Curay, Acting Director, Office of Biotechnology...

  12. UVA Causes Dual Inactivation of Cathepsin B and L Underlying Lysosomal Dysfunction in Human Dermal Fibroblasts

    Science.gov (United States)

    Lamore, Sarah D.; Wondrak, Georg T.

    2013-01-01

    Cutaneous exposure to chronic solar UVA-radiation is a causative factor in photocarcinogenesis and photoaging. Recently, we have identified the thiol-dependent cysteine-protease cathepsin B as a novel UVA-target undergoing photo-oxidative inactivation upstream of autophagic-lysosomal dysfunction in fibroblasts. In this study, we examined UVA effects on a wider range of cathepsins and explored the occurrence of UVA-induced cathepsin inactivation in other cultured skin cell types. In dermal fibroblasts, chronic exposure to non-cytotoxic doses of UVA caused pronounced inactivation of the lysosomal cysteine-proteases cathepsin B and L, effects not observed in primary keratinocytes and occurring only to a minor extent in primary melanocytes. In order to determine if UVA-induced lysosomal impairment requires single or dual inactivation of cathepsin B and/or L, we used a genetic approach (siRNA) to selectively downregulate enzymatic activity of these target cathepsins. Monitoring an established set of protein markers (including LAMP1, LC3-II, and p62) and cell ultrastructural changes detected by electron microscopy, we observed that only dual genetic antagonism (targeting both CTSB and CTSL expression) could mimic UVA-induced autophagic-lysosomal alterations, whereas single knockdown (targeting CTSB or CTSL only) did not display ‘UVA-mimetic’ effects failing to reproduce the UVA-induced phenotype. Taken together, our data demonstrate that chronic UVA inhibits both cathepsin B and L enzymatic activity and that dual inactivation of both enzymes is a causative factor underlying UVA-induced impairment of lysosomal function in dermal fibroblasts. PMID:23603447

  13. Permanent genetic access to transiently active neurons via TRAP: targeted recombination in active populations.

    Science.gov (United States)

    Guenthner, Casey J; Miyamichi, Kazunari; Yang, Helen H; Heller, H Craig; Luo, Liqun

    2013-06-05

    Targeting genetically encoded tools for neural circuit dissection to relevant cellular populations is a major challenge in neurobiology. We developed an approach, targeted recombination in active populations (TRAP), to obtain genetic access to neurons that were activated by defined stimuli. This method utilizes mice in which the tamoxifen-dependent recombinase CreER(T2) is expressed in an activity-dependent manner from the loci of the immediate early genes Arc and Fos. Active cells that express CreER(T2) can only undergo recombination when tamoxifen is present, allowing genetic access to neurons that are active during a time window of less than 12 hr. We show that TRAP can provide selective access to neurons activated by specific somatosensory, visual, and auditory stimuli and by experience in a novel environment. When combined with tools for labeling, tracing, recording, and manipulating neurons, TRAP offers a powerful approach for understanding how the brain processes information and generates behavior. Copyright © 2013 Elsevier Inc. All rights reserved.

  14. Identification and characterization of Clonorchis sinensis cathepsin B proteases in the pathogenesis of clonorchiasis.

    Science.gov (United States)

    Chen, Wenjun; Ning, Dan; Wang, Xiaoyun; Chen, Tingjin; Lv, Xiaoli; Sun, Jiufeng; Wu, De; Huang, Yan; Xu, Jin; Yu, Xinbing

    2015-12-21

    Human clonorchiasis is a prevailing food-borne disease caused by Clonorchis sinensis infection. Functional characterizations of key molecules from C. sinensis could facilitate the intervention of C. sinensis associated diseases. In this study, immunolocalization of C. sinensis cathepsin B proteases (CsCBs) in C. sinensis worms was investigated. Four CsCBs were expressed in Pichia pastoris yeast cells. Purified yCsCBs were measured for enzymatic and hydrolase activities in the presence of various host proteins. Cell proliferation, wound-healing and transwell assays were performed to show the effect of CsCBs on human cells. CsCBs were localized in the excretory vesicle, oral sucker and intestinal tract of C. sinensis. Recombinant yCsCBs from yeast showed active enzymatic activity at pH 5.0-5.5 and at 37-42 °C. yCsCBs can degrade various host proteins including human serum albumin, human fibronectin, human hemoglobin and human IgG. CsCBs were detected in liver tissues of mice and cancer patients afflicted with clonorchiasis. Various bioassays collectively demonstrated that CsCBs could promote cell proliferation, migration and invasion of human cancer cells. Our results demonstrated that CsCBs can degrade various human proteins and we proved that the secreted CsCBs are involved in the pathogenesis of clonorchiasis.

  15. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    International Nuclear Information System (INIS)

    Inami, Yoshihiro; Yamashina, Shunhei; Izumi, Kousuke; Ueno, Takashi; Tanida, Isei; Ikejima, Kenichi; Watanabe, Sumio

    2011-01-01

    Highlights: → Acidification of autophagosome was blunted in steatotic hepatocytes. → Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. → Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. → Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  16. Hepatic steatosis inhibits autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression

    Energy Technology Data Exchange (ETDEWEB)

    Inami, Yoshihiro [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Yamashina, Shunhei, E-mail: syamashi@juntendo.ac.jp [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Izumi, Kousuke [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Ueno, Takashi [Department of Biochemistry, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan); Tanida, Isei [Department of Biochemistry and Cell Biology, Laboratory of Biomembranes, National Institute of Infectious Disease, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640 (Japan); Ikejima, Kenichi; Watanabe, Sumio [Department of Gastroenterology, Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku, Tokyo 113-8421 (Japan)

    2011-09-09

    Highlights: {yields} Acidification of autophagosome was blunted in steatotic hepatocytes. {yields} Hepatic steatosis did not disturb fusion of isolated autophagosome and lysosome. {yields} Proteinase activity of cathepsin B and L in autolysosomes was inhibited by steatosis. {yields} Hepatic expression of cathepsin B and L was suppressed by steatosis. -- Abstract: Autophagy, one of protein degradation system, contributes to maintain cellular homeostasis and cell defense. Recently, some evidences indicated that autophagy and lipid metabolism are interrelated. Here, we demonstrate that hepatic steatosis impairs autophagic proteolysis. Though accumulation of autophagosome is observed in hepatocytes from ob/ob mice, expression of p62 was augmented in liver from ob/ob mice more than control mice. Moreover, degradation of the long-lived protein leucine was significantly suppressed in hepatocytes isolated from ob/ob mice. More than 80% of autophagosomes were stained by LysoTracker Red (LTR) in hepatocytes from control mice; however, rate of LTR-stained autophagosomes in hepatocytes were suppressed in ob/ob mice. On the other hand, clearance of autolysosomes loaded with LTR was blunted in hepatocytes from ob/ob mice. Although fusion of isolated autophagosome and lysosome was not disturbed, proteinase activity of cathepsin B and L in autolysosomes and cathepsin B and L expression of liver were suppressed in ob/ob mice. These results indicate that lipid accumulation blunts autophagic proteolysis via impairment of autophagosomal acidification and cathepsin expression.

  17. Modulation of cathepsin G expression in severe atopic dermatitis following medium-dose UVA1 phototherapy

    Directory of Open Access Journals (Sweden)

    Altmeyer Peter

    2002-08-01

    Full Text Available Abstract Background During the last decade, medium-dose UVA1 phototherapy (50 J/cm2 has achieved great value within the treatment of severe atopic dermatitis (AD. The purpose of our study was to investigate to what extent UVA1 irradiation is able to modulate the status of protease activity by the use of a monoclonal antibody labeling cathepsin G. Methods In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement of skin status in patients with AD, biopsy specimens from 15 patients before and after treatment were analyzed immunohistochemically for proteolytic activation. Results Compared to lesional skin of patients with AD before UVA1 irradiation, the number of cells positive for cathepsin G within the dermal infiltrate decreased significantly after treatment. The decrease of cathepsin G+ cells was closely linked to a substantial clinical improvement in skin condition. Conclusions In summary, our findings demonstrated that medium-dose UVA1 irradiation leads to a modulation of the expression of cathepsin G in the dermal inflammatory infiltrate in patients with severe AD. Cathepsin G may attack laminin, proteoglycans, collagen I and insoluble fibronectin, to provoke proinflammatory events, to degrade the basement membrane, to destroy the tissue inhibitor of metalloproteinases and to increase the endothelial permeability. Therefore, its down-regulation by UVA1 phototherapy may induce the reduction of skin inflammation as well as improvement of the skin condition.

  18. Modulation of cathepsin G expression in severe atopic dermatitis following medium-dose UVA1 phototherapy

    Science.gov (United States)

    Breuckmann, Frank; von Kobyletzki, Gregor; Avermaete, Annelies; Kreuter, Alexander; Altmeyer, Peter; Gambichler, Thilo

    2002-01-01

    Background During the last decade, medium-dose UVA1 phototherapy (50 J/cm2) has achieved great value within the treatment of severe atopic dermatitis (AD). The purpose of our study was to investigate to what extent UVA1 irradiation is able to modulate the status of protease activity by the use of a monoclonal antibody labeling cathepsin G. Methods In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement of skin status in patients with AD, biopsy specimens from 15 patients before and after treatment were analyzed immunohistochemically for proteolytic activation. Results Compared to lesional skin of patients with AD before UVA1 irradiation, the number of cells positive for cathepsin G within the dermal infiltrate decreased significantly after treatment. The decrease of cathepsin G+ cells was closely linked to a substantial clinical improvement in skin condition. Conclusions In summary, our findings demonstrated that medium-dose UVA1 irradiation leads to a modulation of the expression of cathepsin G in the dermal inflammatory infiltrate in patients with severe AD. Cathepsin G may attack laminin, proteoglycans, collagen I and insoluble fibronectin, to provoke proinflammatory events, to degrade the basement membrane, to destroy the tissue inhibitor of metalloproteinases and to increase the endothelial permeability. Therefore, its down-regulation by UVA1 phototherapy may induce the reduction of skin inflammation as well as improvement of the skin condition. PMID:12204095

  19. Identification and characterization of the novel reversible and selective cathepsin X inhibitors.

    Science.gov (United States)

    Fonović, Urša Pečar; Mitrović, Ana; Knez, Damijan; Jakoš, Tanja; Pišlar, Anja; Brus, Boris; Doljak, Bojan; Stojan, Jure; Žakelj, Simon; Trontelj, Jurij; Gobec, Stanislav; Kos, Janko

    2017-09-13

    Cathepsin X is a cysteine peptidase involved in the progression of cancer and neurodegenerative diseases. Targeting this enzyme with selective inhibitors opens a new possibility for intervention in several therapeutic areas. In this study triazole-based reversible and selective inhibitors of cathepsin X have been identified. Their selectivity and binding is enhanced when the 2,3-dihydrobenzo[b][1,4]dioxine moiety is present as the R 1 substituent. Of a series of selected triazole-benzodioxine derivatives, compound 22 is the most potent inhibitor of cathepsin X carboxypeptidase activity (K i  = 2.45 ± 0.05 μM) with at least 100-fold greater selectivity in comparison to cathepsin B or other related cysteine peptidases. Compound 22 is not cytotoxic to prostate cancer cells PC-3 or pheochromocytoma PC-12 cells at concentrations up to 10 μM. It significantly inhibits the migration of tumor cells and increases the outgrowth of neurites, both processes being under the control of cathepsin X carboxypeptidase activity. Compound 22 and other characterized triazole-based inhibitors thus possess a great potential for further development resulting in several in vivo applications.

  20. A recombinant Anticarsia gemmatalis MNPV harboring chiA and v-cath genes from Choristoneura fumiferana defective NPV induce host liquefaction and increased insecticidal activity.

    Directory of Open Access Journals (Sweden)

    Anabele Azevedo Lima

    Full Text Available One of the interesting features of Anticarsia gemmatalis multiple nucleopolyhedrovirus isolate 2D (AgMNPV-2D genome is the absence of chitinase (chiA and cathepsin (v-cath genes. This characteristic may be responsible for the lack of liquefaction and melanization in A. gemmatalis larvae killed by AgMNPV-2D infection. This study aimed to test the hypothesis that CHIA and V-CATH proteins from Choristonera fumiferana DEF multiple nucleopolyhedrovirus (CfDEFNPV are able to liquefy and melanize the cuticle of A. gemmatalis larvae infected by a recombinant AgMNPV containing chiA and v-cath genes inserted in its genome. A fragment from the CfDefNPV genome containing chiA and v-cath genes was inserted into the genome of AgMNPV-2D. The recombinant virus (vAgp2100Cf.chiA/v-cath was purified and used to infect insect cells and larvae. Transcripts of v-cath and chiA genes were detected along the infection of insect cells by qRT-PCR, from early to late phases of infection. The analysis of A. gemmatalis larvae killed by vAgp2100Cf.chiA/v-cath infection confirmed the hypothesis proposed. The vAgp2100Cf.chiA/v-cath showed higher insecticidal activity against third instar A. gemmatalis larvae when compared to AgMNPV-2D. The mean time to death was also lower for the vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D at 10 days post infection. Occlusion body production was higher in A. gemmatalis larvae infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. Enzyme assays showed higher chitinase and cysteine protease activities in insect cells and insects infected with vAgp2100Cf.chiA/v-cath when compared to AgMNPV-2D. The introduction of chiA and v-cath genes into the genome of AgMNPV improves its insecticidal activity against A. gemmatalis larvae and this recombinant virus could be used as an alternative to the wild type virus to control this important insect pest.

  1. Inhibitory assay for degradation of collagen IV by cathepsin B with a surface plasmon resonance sensor.

    Science.gov (United States)

    Shoji, Atsushi; Suenaga, Yumiko; Hosaka, Atsushi; Ishida, Yuuki; Yanagida, Akio; Sugawara, Masao

    2017-10-25

    We describe a simple method for evaluating the inhibition of collagen IV degradation by cathepsin B with a surface plasmon resonance (SPR) biosensor. The change in the SPR signal decreased with an increase in the concentration of cathepsin B inhibitors. The order of the inhibitory constant (Ki) obtained by the SPR method was CA074Me≈Z-Phe-Phe-FMK < leupeptin. This order was different from that obtained by benzyloxycarbonyl-Phe-Phe-Fluoromethylketone (Z-Phe-Phe-FMK) as a peptide substrate. The comparison of Ki suggested that CA074 and Z-Phe-Phe-FMK inhibited exopeptidase activity, and leupeptin inhibited the endopeptidase activity of cathepsin B more strongly. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. EKSTRAKSI DAN KARAKTERISASI PARSIAL EKSTRAK KASAR ENZIM KATEPSIN DARI IKAN PATIN [Extraction and Partial Characterization of Crude Enzymes Cathepsin from Catfish

    Directory of Open Access Journals (Sweden)

    Muhammad Zakiyul Fikri*

    2014-06-01

    Full Text Available Decomposition of protein by enzymatic process will lead to changes in odor, texture, and appearance of fish. The enzymes that play a role in the enzymatic process is primarily proteolytic enzymes. Cathepsin is one of the proteolytic enzymes found in animal tissue that hydrolyzes peptide bonds of proteins. This study aims to extract the cathepsin, characterize the crude extract derived from catfish. The stages of this research consist of the extraction and characterization of the cathepsin from catfish. Result of the extraction was crude extract of cathepsin with activity of 0.278 U/mL. The enzyme had optimum temperature of 50°C, pH 6 and substrate concentration of 2%. The activity of the cathepsin was inhibited by metal ions of Fe3+, Cu2+, Ca2+, but increased by metal ions of Mg2+.

  3. A cathepsin F-like peptidase involved in barley grain protein mobilization, HvPap-1, is modulated by its own propeptide and by cystatins

    Science.gov (United States)

    Diaz, Isabel

    2012-01-01

    Among the C1A cysteine proteases, the plant cathepsin F-like group has been poorly studied. This paper describes the molecular and functional characterization of the HvPap-1 cathepsin F-like protein from barley. This peptidase is N-glycosylated and has to be processed to become active by its own propeptide being an important modulator of the peptidase activity. The expression pattern of its mRNA and protein suggest that it is involved in different proteolytic processes in the barley plant. HvPap-1 peptidase has been purified in Escherichia coli and the recombinant protein is able to degrade different substrates, including barley grain proteins (hordeins, albumins, and globulins) stored in the barley endosperm. It has been localized in protein bodies and vesicles of the embryo and it is induced in aleurones by gibberellin treatment. These three features support the implication of HvPap-1 in storage protein mobilization during grain germination. In addition, a complex regulation exerted by the barley cystatins, which are cysteine protease inhibitors, and by its own propeptide, is also described PMID:22791822

  4. Cathepsin K deficiency in mice induces structural and metabolic changes in the central nervous system that are associated with learning and memory deficits

    Directory of Open Access Journals (Sweden)

    Oswald Julia

    2011-07-01

    Full Text Available Abstract Background Cathepsin K is a cysteine peptidase known for its importance in osteoclast-mediated bone resorption. Inhibitors of cathepsin K are in clinical trials for treatment of osteoporosis. However, side effects of first generation inhibitors included altered levels of related cathepsins in peripheral organs and in the central nervous system (CNS. Cathepsin K has been recently detected in brain parenchyma and it has been linked to neurobehavioral disorders such as schizophrenia. Thus, the study of the functions that cathepsin K fulfils in the brain becomes highly relevant. Results Cathepsin K messenger RNA was detectable in all brain regions of wild type (WT mice. At the protein level, cathepsin K was detected by immunofluorescence microscopy in vesicles of neuronal and non-neuronal cells throughout the mouse brain. The hippocampus of WT mice exhibited the highest levels of cathepsin K activity in fluorogenic assays, while the cortex, striatum, and cerebellum revealed significantly lower enzymatic activities. At the molecular level, the proteolytic network of cysteine cathepsins was disrupted in the brain of cathepsin K-deficient (Ctsk-/- animals. Specifically, cathepsin B and L protein and activity levels were altered, whereas cathepsin D remained largely unaffected. Cystatin C, an endogenous inhibitor of cysteine cathepsins, was elevated in the striatum and hippocampus, pointing to regional differences in the tissue response to Ctsk ablation. Decreased levels of astrocytic glial fibrillary acidic protein, fewer and less ramified profiles of astrocyte processes, differentially altered levels of oligodendrocytic cyclic nucleotide phosphodiesterase, as well as alterations in the patterning of neuronal cell layers were observed in the hippocampus of Ctsk-/- mice. A number of molecular and cellular changes were detected in other brain regions, including the cortex, striatum/mesencephalon, and cerebellum. Moreover, an overall induction of

  5. Recombinant dioscorins of the yam storage protein expressed in Escherichia coli exhibit antioxidant and immunomodulatory activities.

    Science.gov (United States)

    Jheng, Yi-Jyun; Tsai, Wei-Yi; Chen, Kuo-Hsuan; Lin, Kuo-Wei; Chyan, Chia-Lin; Yang, Ching-Chi; Lin, Kuo-Chih

    2012-09-01

    Dioscorins, the major storage proteins in yam tubers, exhibit biochemical and immunomodulatroy activities. To investigate the potential application of dioscorins in biomedical research, we expressed the dioscorin genes Dj-dioA3 and Dp-dioA2 from Dioscorea japonica and Dioscorea pseudojaponica, respectively, in E. coli and routinely obtained approximately 15 mg proteins per liter Escherichia coli culture (mg/L) to 30 mg/L of rDj-dioscorinA3 and 4 to 8 mg/L of rDp-dioscorinA2. Western blot analyses revealed that both recombinant dioscorins contained epitopes with similar antigenicities to those of the native dioscorins. Results from dithiothreitol (DTT) treatment followed by monobromobimane (mBBr) staining showed that both recombinant dioscorins, like the native dioscorins, contain an intramolecular disulfide bond between Cys(28) and Cys(187) residues. Circular dichroism spectroscopy findings indicated that the secondary structural contents of the recombinant dioscorins showed high similarity to those of their corresponding native dioscorins. Both recombinant dioscorins, like the native dioscorins, exhibited 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging and Toll-like receptor 4 signaling activities, and stimulated the phagocytosis of E. coli by macrophage. Overall, our results indicated that substantial amounts of recombinant dioscorins can be purified easily from E. coli and that these recombinant dioscorins are appropriate for application in future investigations of the biomedical functions of dioscorins. Copyright © 2012 Elsevier Inc. All rights reserved.

  6. A novel nonsense mutation in cathepsin C gene in an Egyptian ...

    African Journals Online (AJOL)

    Background: Cathepsin C gene (CTSC) (MIM#602365) is a lysosomal cysteine proteinase coding gene which encodes for CTSC protein that plays a major role in the activation of granule serine proteases, particularly leukocyte elastase and granzymes A and B. This activity was proposed to play a role in epithelial ...

  7. A broad survey of cathepsin K immunoreactivity in human neoplasms.

    Science.gov (United States)

    Zheng, Gang; Martignoni, Guido; Antonescu, Cristina; Montgomery, Elizabeth; Eberhart, Charles; Netto, George; Taube, Janis; Westra, William; Epstein, Jonathan I; Lotan, Tamara; Maitra, Anirban; Gabrielson, Edward; Torbenson, Michael; Iacobuzio-Donahue, Christine; Demarzo, Angelo; Shih, Ie Ming; Illei, Peter; Wu, T C; Argani, Pedram

    2013-02-01

    Cathepsin K is consistently and diffusely expressed in alveolar soft part sarcoma (ASPS) and a subset of translocation renal cell carcinomas (RCCs). However, cathepsin K expression in human neoplasms has not been systematically analyzed. We constructed tissue microarrays (TMA) from a wide variety of human neoplasms, and performed cathepsin K immunohistochemistry (IHC). Only 2.7% of 1,140 carcinomas from various sites exhibited cathepsin K labeling, thus suggesting that among carcinomas, cathepsin K labeling is highly specific for translocation RCC. In contrast to carcinomas, cathepsin K labeling was relatively common (54.6%) in the 414 mesenchymal lesions studied, including granular cell tumor, melanoma, and histiocytic lesions, but not paraganglioma, all of which are in the morphologic differential diagnosis of ASPS. Cathepsin K IHC can be helpful in distinguishing ASPS and translocation RCC from some but not all of the lesions in their differential diagnosis.

  8. 75 FR 21008 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Actions Under the NIH...

    Science.gov (United States)

    2010-04-22

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology... Biotechnology Activities (OBA) published a proposal to revise the NIH Guidelines for Research with Recombinant... by fax to 301-496-9839 or mail to the Office of Biotechnology Activities, National Institutes of...

  9. Transient expression of progesterone receptor and cathepsin-l in human granulosa cells during the periovulatory period.

    Science.gov (United States)

    García, Víctor; Kohen, Paulina; Maldonado, Carola; Sierralta, Walter; Muñoz, Alex; Villarroel, Claudio; Strauss, Jerome F; Devoto, Luigi

    2012-03-01

    To study in vivo the progesterone receptor (PR) expression levels in human granulosa cells (GCs) during the periovulatory period and the affect of the protein kinase A (PKA) pathway on PR expression and cathepsin-L expression-activation. Experimental study. University research unit. Twenty-five women of reproductive age. Follicular fluid and GCs obtained from spontaneous cycles before and during the normal luteinizing hormone surge, and samples obtained 36 hours after human chorionic gonadotropin (hCG) administration in patients undergoing in vitro fertilization. To determine PR, cathepsin-L messenger RNA (mRNA) analysis via real-time polymerase chain reaction, and protein of PR, cathepsin-L, and PKA in human GCs. The Western blot analysis revealed that bands of PR (isoform A) were the most abundant and that mRNA (PR-A and PR-B) have a temporal pattern of expression throughout the periovulatory period. The protein levels of PR and cathepsin-L were up-regulated by hCG. The abundance of PR was diminished in the presence of PKA inhibitor, and cathepsin-L with PR receptor antagonist. The transient expression of PR in human GCs of the preovulatory follicle suggests that PR and its ligand play a role in the activation of cathepsin-L, which is presumably involved in the degradation of the follicular extracellular matrix during human ovulation. Copyright © 2012 American Society for Reproductive Medicine. All rights reserved.

  10. Characterization of DNA binding, transcriptional activation, and regulated nuclear association of recombinant human NFATp

    Directory of Open Access Journals (Sweden)

    Seto Anita G

    2000-11-01

    Full Text Available Abstract Background NFATp is one member of a family of transcriptional activators whose nuclear accumulation and hence transcriptional activity is regulated in mammalian cells. Human NFATp exists as a phosphoprotein in the cytoplasm of naive T cells. Upon antigen stimulation, NFATp is dephosphorylated, accumulates in nuclei, and functions to regulate transcription of genes including those encoding cytokines. While the properties of the DNA binding domain of NFATp have been investigated in detail, biochemical studies of the transcriptional activation and regulated association with nuclei have remained unexplored because of a lack of full length, purified recombinant NFATp. Results We developed methods for expressing and purifying full length recombinant human NFATp that has all of the properties known to be associated with native NFATp. The recombinant NFATp binds DNA on its own and cooperatively with AP-1 proteins, activates transcription in vitro, is phosphorylated, can be dephosphorylated by calcineurin, and exhibits regulated association with nuclei in vitro. Importantly, activation by recombinant NFATp in a reconstituted transcription system required regions of the protein outside of the central DNA binding domain. Conclusions We conclude that NFATp is a bona fide transcriptional activator. Moreover, the reagents and methods that we developed will facilitate future studies on the mechanisms of transcriptional activation and nuclear accumulation by NFATp, a member of an important family of transcriptional regulatory proteins.

  11. Antiproliferative activity of recombinant human interferon-λ2 ...

    African Journals Online (AJOL)

    Jane

    2011-07-20

    Jul 20, 2011 ... antiviral activity, antiproliferative activity and in vivo antitumor activity .... bovine serum albumin (BSA) and 0.05% (v/v) Tween 20 in PBS at room temperature for 1 h .... identified and developed into an important insect embryo.

  12. Crystal structure of cathepsin A, a novel target for the treatment of cardiovascular diseases

    Energy Technology Data Exchange (ETDEWEB)

    Schreuder, Herman A., E-mail: herman.schreuder@sanofi.com; Liesum, Alexander, E-mail: alexander.liesum@sanofi.com; Kroll, Katja, E-mail: katja.kroll@sanofi.com; Böhnisch, Britta, E-mail: britta.boehnisch@sanofi.com; Buning, Christian, E-mail: christian.buning@sanofi.com; Ruf, Sven, E-mail: sven.ruf@sanofi.com; Sadowski, Thorsten, E-mail: thorsten.sadowski@sanofi.com

    2014-03-07

    Graphical abstract: - Highlights: • The structures of active cathepsin A and the inactive precursor are very similar. • The only major difference is the absence of a 40 residue activation domain. • The termini of the active catalytic core are held together by a disulfide bond. • Compound 1 reacts with the catalytic Ser150, building a tetrahedral intermediate. • Compound 2 is cleaved by the enzyme and a fragment remained bound. - Abstract: The lysosomal serine carboxypeptidase cathepsin A is involved in the breakdown of peptide hormones like endothelin and bradykinin. Recent pharmacological studies with cathepsin A inhibitors in rodents showed a remarkable reduction in cardiac hypertrophy and atrial fibrillation, making cathepsin A a promising target for the treatment of heart failure. Here we describe the crystal structures of activated cathepsin A without inhibitor and with two compounds that mimic the tetrahedral intermediate and the reaction product, respectively. The structure of activated cathepsin A turned out to be very similar to the structure of the inactive precursor. The only difference was the removal of a 40 residue activation domain, partially due to proteolytic removal of the activation peptide, and partially by an order–disorder transition of the peptides flanking the removed activation peptide. The termini of the catalytic core are held together by the Cys253–Cys303 disulfide bond, just before and after the activation domain. One of the compounds we soaked in our crystals reacted covalently with the catalytic Ser150 and formed a tetrahedral intermediate. The other compound got cleaved by the enzyme and a fragment, resembling one of the natural reaction products, was found in the active site. These studies establish cathepsin A as a classical serine proteinase with a well-defined oxyanion hole. The carboxylate group of the cleavage product is bound by a hydrogen-bonding network involving one aspartate and two glutamate side chains

  13. Effect of haemodilution, acidosis, and hypothermia on the activity of recombinant factor VIIa (NovoSeven)

    DEFF Research Database (Denmark)

    Viuff, D.; Lauritzen, B.; Pusateri, A.E.

    2008-01-01

    BACKGROUND: A range of plasma volume expanders is used clinically, often in settings where haemostasis may already be impaired. The haemostatic agent, recombinant activated factor VII (rFVIIa, NovoSeven), may be used to improve haemostasis but potential interactions with different volume expanders...

  14. Efficacy and safety of recombinant activated factor VII for acute intracerebral hemorrhage

    DEFF Research Database (Denmark)

    Mayer, Stephan A; Brun, Nikolai C; Begtrup, Kamilla

    2008-01-01

    BACKGROUND: Intracerebral hemorrhage is the least treatable form of stroke. We performed this phase 3 trial to confirm a previous study in which recombinant activated factor VII (rFVIIa) reduced growth of the hematoma and improved survival and functional outcomes. METHODS: We randomly assigned 841...

  15. Effect of copper on the recombination activity of extended defects in silicon

    Energy Technology Data Exchange (ETDEWEB)

    Feklisova, O. V., E-mail: feklisov@iptm.ru; Yakimov, E. B. [Russian Academy of Sciences, Institute of Microelectronics Technology and High-Purity Materials (Russian Federation)

    2015-06-15

    The effect of copper atoms introduced by high-temperature diffusion on the recombination properties of dislocations and dislocation trails in p-type single-crystal silicon is studied by the electron-beam-induced current technique. It is shown that, in contrast to dislocations, dislocation trails exhibit an increase in recombination activity after the introduction of copper. Bright contrast appearance in the vicinity of dislocation trails is detected after the diffusion of copper and quenching of the samples. The contrast depends on the defect density in these trails.

  16. Anti-angiogenesis and anti-tumor activity of recombinant anginex

    International Nuclear Information System (INIS)

    Brandwijk, Ricardo J.M.G.E.; Dings, Ruud P.M.; Linden, Edith van der; Mayo, Kevin H.; Thijssen, Victor L.J.L.; Griffioen, Arjan W.

    2006-01-01

    Anginex, a synthetic 33-mer angiostatic peptide, specifically inhibits vascular endothelial cell proliferation and migration along with induction of apoptosis in endothelial cells. Here we report on the in vivo characterization of recombinant anginex and use of the artificial anginex gene for gene therapy approaches. Tumor growth of human MA148 ovarian carcinoma in athymic mice was inhibited by 80% when treated with recombinant anginex. Histological analysis of the tumors showed an approximate 2.5-fold reduction of microvessel density, suggesting that angiogenesis inhibition is the cause of the anti-tumor effect. Furthermore, there was a significant correlation between the gene expression patterns of 16 angiogenesis-related factors after treatment with both recombinant and synthetic anginex. To validate the applicability of the anginex gene for gene therapy, stable transfectants of murine B16F10 melanoma cells expressing recombinant anginex were made. Supernatants of these cells inhibited endothelial cell proliferation in vitro. Furthermore, after subcutaneous injection of these cells in C57BL/6 mice, an extensive delay in tumor growth was observed. These data show that the artificial anginex gene can be used to produce a recombinant protein with similar activity as its synthetic counterpart and that the gene can be applied in gene therapy approaches for cancer treatment

  17. High-Level Expression of Recombinant Bovine Lactoferrin in Pichia pastoris with Antimicrobial Activity

    Directory of Open Access Journals (Sweden)

    Blanca Iglesias-Figueroa

    2016-06-01

    Full Text Available In this study, bovine lactoferrin (bLf, an iron-binding glycoprotein considered an important nutraceutical protein because of its several properties, was expressed in Pichia pastoris KM71-H under AOX1 promoter control, using pJ902 as the recombinant plasmid. Dot blotting analysis revealed the expression of recombinant bovine lactoferrin (rbLf in Pichia pastoris. After Bach fermentation and purification by molecular exclusion, we obtained an expression yield of 3.5 g/L of rbLf. rbLf and predominantly pepsin-digested rbLf (rbLfcin demonstrated antibacterial activity against Escherichia coli (E. coli BL21DE3, Staphylococcus aureus (S. aureus FRI137, and, in a smaller percentage, Pseudomonas aeruginosa (Ps. Aeruginosa ATCC 27833. The successful expression and characterization of functional rbLf expressed in Pichia pastoris opens a prospect for the development of natural antimicrobial agents produced recombinantly.

  18. Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies.

    Science.gov (United States)

    Yamaguchi, Hiroshi; Miyazaki, Masaya

    2014-02-20

    Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  19. Analysis of heparanase isoforms and cathepsin B in the plasma of patients with gastrointestinal carcinomas: analytical cross-sectional study

    Directory of Open Access Journals (Sweden)

    Carina Mucciolo Melo

    Full Text Available CONTEXT AND OBJECTIVE: Heparanase-1 degrades heparan sulfate and has been correlated with tumor progression. Although the isoform heparanase-2 has no catalytic activity, it seems to be important for modulating heparanase-1 activity. Cathepsin B is a proteinase involved in tumor metastasis. The aim of this study was to analyze heparanase isoform expression and cathepsin B activity in plasma samples from patients with gastrointestinal carcinomas, compared with healthy individuals (control group. DESIGN AND SETTING: This was an analytical cross-sectional study. Peripheral blood samples were collected at a Brazilian public hospital, from 21 patients with histopathological diagnoses of gastrointestinal carcinomas and from 43 healthy individuals. The analyses were performed in two Brazilian medical schools. METHODS: Heparanase isoforms were identified and quantified in plasma samples by means of Western blot. The enzymatic activities of heparanase-1 and cathepsin B were also measured. RESULTS: The results demonstrated that the expression of both heparanase isoforms was significantly greater in plasma samples from gastrointestinal carcinoma patients, compared with the control group. Logistic regression analysis showed that increased heparanase-1 and heparanase-2 expression was exclusively dependent on the tumor. There was a significant increase in heparanase-1 and cathepsin B activity in the patients' plasma. CONCLUSION: Overexpression of heparanase-1 and heparanase-2, along with increased heparanase-1 and cathepsin B activity in plasma, is associated with the diagnosis of gastrointestinal carcinoma. These findings provide support for using non-invasive assays (plasma samples as an auxiliary method for diagnosing gastrointestinal tumors.

  20. Cathepsin B is involved in the heat shock induced cardiomyocytes apoptosis as well as the anti-apoptosis effect of HSP-70.

    Science.gov (United States)

    Hsu, Shu-Fen; Hsu, Chuan-Chih; Cheng, Bor-Chih; Lin, Cheng-Hsien

    2014-11-01

    Cathepsin B is one of the major lysosomal cysteine proteases that plays an important role in apoptosis. Herein, we investigated whether Cathepsin B is involved in cardiomyocyte apoptosis caused by hyperthermic injury (HI) and heat shock protein (HSP)-70 protects these cells from HI-induced apoptosis mediated by Cathepsin. HI was produced in H9C2 cells by putting them in a circulating 43 °C water bath for 120 min, whereas preinduction of HSP-70 was produced in H9C2 cells by mild heat preconditioning (or putting them in 42 °C water bath for 30 min) 8 h before the start of HI. It was found that HI caused both cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. E-64-c, in addition to reducing Cathepsin B activity, significantly attenuated HI-induced cardiomyocyte apoptosis (evidenced by increased apoptotic cell numbers, increased tuncated Bid (t-Bid), increased cytochrome C, increased caspase-9/-3, and decreased Bcl-2/Bax) in H9C2 cells. In addition, preinduction of HSP-70 by mild heat preconditioning or inhibition of HSP-70 by Tripolide significantly attenuated or exacerbated respectively both the cardiomyocyte apoptosis and increased Cathepsin B activity in H9C2 cells. Furthermore, the beneficial effects of pre-induction of HSP-70 by mild heat production in reducing both cardiomyocyte apoptosis and increased Cathepsin B activity caused by HI can be significantly reduced by Triptolide preconditioning. These results indicate that Cathepsin B is involved in HI-induced cardiomyocyte apoptosis in H9C2 cells and HSP-70 protects these cells from HI-induced cardiomyocyte apoptosis through Cathepsin B pathways.

  1. Delivery of rSLPI in a liposomal carrier for inhalation provides protection against cathepsin L degradation

    OpenAIRE

    Gibbons, Aileen M.; McElvaney, Noel; Taggart, Clifford; Cryan, Sally-Ann

    2009-01-01

    Secretory leukocyte protease inhibitor (SLPI) is an endogenous serine protease inhibitor that protects the lungs from excessive tissue damage caused by leukocyte proteases released during inflammation. Recombinant SLPI (rSLPI) has shown potential as a treatment for inflammatory lung conditions. To date, its clinical application has been limited by rapid enzymatic cleavage by cathepsins and rapid clearance from the lungs after inhalation. In this study, rSLPI was encapsulated in 1,2-Dioleoyl-s...

  2. Antifibrotic effects of curcumin are associated with overexpression of cathepsins K and L in bleomycin treated mice and human fibroblasts

    Directory of Open Access Journals (Sweden)

    Zhang Dongwei

    2011-11-01

    Full Text Available Abstract Background Lung fibrosis is characterized by fibroblast proliferation and the deposition of collagens. Curcumin, a polyphenol antioxidant from the spice tumeric, has been shown to effectively counteract fibroblast proliferation and reducing inflammation and fibrotic progression in animal models of bleomycin-induced lung injury. However, there is little mechanistic insight in the biological activity of curcumin. Here, we study the effects of curcumin on the expression and activity of cathepsins which have been implicated in the development of fibrotic lung diseases. Methods We investigated the effects of curcumin administration to bleomycin stimulated C57BL/6 mice and human fetal lung fibroblasts (HFL-1 on the expression of cathepsins K and L which have been implicated in matrix degradation, TGF-β1 modulation, and apoptosis. Lung tissues were evaluated for their contents of cathepsins K and L, collagen, and TGF-β1. HFL-1 cells were used to investigate the effects of curcumin and cathepsin inhibition on cell proliferation, migration, apoptosis, and the expression of cathepsins K and L and TGF-β1. Results Collagen deposition in lungs was decreased by 17-28% after curcumin treatment which was accompanied by increased expression levels of cathepsins L (25%-39% and K (41%-76% and a 30% decrease in TGF-β1 expression. Moreover, Tunel staining of lung tissue revealed a 33-41% increase in apoptotic cells after curcumin treatment. These in vivo data correlated well with data obtained from the human fibroblast line, HFL-1. Here, cathepsin K and L expression increased 190% and 240%, respectively, in the presence of curcumin and the expression of TGF-β1 decreased by 34%. Furthermore, curcumin significantly decreased cell proliferation and migration and increased the expression of surrogate markers of apoptosis. In contrast, these curcumin effects were partly reversed by a potent cathepsin inhibitor. Conclusion This study demonstrates that

  3. Cysteine and aspartic proteases cathepsins B and D determine the invasiveness of MCF10A neoT cells

    International Nuclear Information System (INIS)

    Premzl, J.; Kos, J.

    2003-01-01

    Background. Lysosomal cathepsins B and D have been reported to play a role in various processes leading to progression of malignant disease. In ras-transformed MCF10A neoT cells both enzymes show similar vesicular distribution in perinuclear and peripheral cytoplasmic regions. Results. The co-localization of cathepsins B and D in some vesicles as defined by confocal microscopy supports their co-ordinate activity in the proteolytic cascade. On the other hand, we showed that stefin A, an endogenous intracellular inhibitor of cysteine proteases, did not co-localize with cathepsin B and is presumably not involved in regulation of its enzymatic activity within the vesicles. Intracellular localization of both enzymes was confined to similar vesicles as the fluorescent degradation products of DQ-collagen IV either in individual cells or cell spheroids. The capability of these two enzymes to degrade collagen and other components of extracellular matrix is further supported by the results of Matrigel invasion assay. We showed that specific intracellular (CA-074 Me) and extracellular (CA-074) inhibitors of cathepsin B and pepstatin A, an inhibitor of cathepsin D, significantly reduced invasion of MCF10A neoT cells. Our results also show that in contrast to some other studies the activation peptide of pro-cathepsin D exhibited no mitogenic effect on MCF10A neoT, MCF-7 or HEK-293 cells. Conclusion. We conclude that lysosomal cysteine proteases cathepsins B and D predominantly participate in degradation of extracellular matrix and facilitate invasion of tumour cells. (author)

  4. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation

    Energy Technology Data Exchange (ETDEWEB)

    Goyal, Shruti; Amar, Saroj Kumar [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow (India); Dubey, Divya; Pal, Manish Kumar [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Singh, Jyoti [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Academy of Scientific and Innovative Research, CSIR-IITR Campus, Lucknow (India); Verma, Ankit; Kushwaha, Hari Narayan [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India); Ray, Ratan Singh, E-mail: ratanray.2011@rediffmail.com [Photobiology Division, CSIR – Indian Institute of Toxicology Research, Post Box No. 80, M.G. Marg, Lucknow 226001, Uttar Pradesh (India)

    2015-12-30

    Highlights: • Photodegradation and formation of photoproduct. • Involvement of ROS in PPD phototoxicity. • Role of ROS in DNA damage, CPD and micronuclei formation. • PPD induced lysosomal destabilization and release of cathepsin B. • Cleavage of Bid and activation of mitochondrial apoptosis. - Abstract: Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings.

  5. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation

    International Nuclear Information System (INIS)

    Goyal, Shruti; Amar, Saroj Kumar; Dubey, Divya; Pal, Manish Kumar; Singh, Jyoti; Verma, Ankit; Kushwaha, Hari Narayan; Ray, Ratan Singh

    2015-01-01

    Highlights: • Photodegradation and formation of photoproduct. • Involvement of ROS in PPD phototoxicity. • Role of ROS in DNA damage, CPD and micronuclei formation. • PPD induced lysosomal destabilization and release of cathepsin B. • Cleavage of Bid and activation of mitochondrial apoptosis. - Abstract: Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings.

  6. Cathepsin D SNP associated with increased risk of variant Creutzfeldt-Jakob disease

    Directory of Open Access Journals (Sweden)

    Sanchez-Juan Pascual

    2008-04-01

    Full Text Available Abstract Background Variant Creutzfeldt-Jakob disease (vCJD originally resulted from the consumption of foodstuffs contaminated by bovine spongiform encephalopathy (BSE material, with 163 confirmed cases in the UK to date. Many thousands are likely to have been exposed to dietary infection and so it is important (for surveillance, epidemic modelling, public health and understanding pathogenesis to identify genetic factors that may affect individual susceptibility to infection. This study looked at a polymorphism in the cathepsin D gene (refSNP ID: rs17571 previously examined in Alzheimer's disease (AD. Methods Blood samples taken from 110 vCJD patients were tested for the C-T base change, and genotype data were compared with published frequencies for a control population using multiple logistic regression. Results There was a significant excess of the cathepsin D polymorphism TT genotype in the vCJD cohort compared to controls. The TT genotype was found to have a 9.75 fold increase in risk of vCJD compared to the CT genotype and a 10.92 fold increase compared to the CC genotype. Conclusion This mutation event has been observed to alter the protease activity of the cathepsin D protein and has been linked to an increase in amyloid beta plaque formation in AD. vCJD neuropathology is characterised by the presence of amyloid plaques, formed from the prion protein, and therefore alterations in the amyloid processing activity of cathepsin D may affect the neuropathogenesis of this disease.

  7. Topical application of recombinant activated factor VII during cesarean delivery for placenta previa.

    Science.gov (United States)

    Schjoldager, Birgit T B G; Mikkelsen, Emmeli; Lykke, Malene R; Præst, Jørgen; Hvas, Anne-Mette; Heslet, Lars; Secher, Niels J; Salvig, Jannie D; Uldbjerg, Niels

    2017-06-01

    During cesarean delivery in patients with placenta previa, hemorrhaging after removal of the placenta is often challenging. In this condition, the extraordinarily high concentration of tissue factor at the placenta site may constitute a principle of treatment as it activates coagulation very effectively. The presumption, however, is that tissue factor is bound to activated factor VII. We hypothesized that topical application of recombinant activated factor VII at the placenta site reduces bleeding without affecting intravascular coagulation. We included 5 cases with planned cesarean delivery for placenta previa. After removal of the placenta, the surgeon applied a swab soaked in recombinant activated factor VII containing saline (1 mg in 246 mL) to the placenta site for 2 minutes; this treatment was repeated once if the bleeding did not decrease sufficiently. We documented the treatment on video recordings and measured blood loss. Furthermore, we determined hemoglobin concentration, platelet count, international normalized ratio, activated partial thrombin time, fibrinogen (functional), factor VII:clot, and thrombin generation in peripheral blood prior to and 15 minutes after removal of the placenta. We also tested these blood coagulation variables in 5 women with cesarean delivery planned for other reasons. Mann-Whitney test was used for unpaired data. In all 5 cases, the uterotomy was closed under practically dry conditions and the median blood loss was 490 (range 300-800) mL. There were no adverse effects of recombinant activated factor VII and we did not measure factor VII to enter the circulation. Neither did we observe changes in thrombin generation, fibrinogen, activated partial thrombin time, international normalized ratio, and platelet count in the peripheral circulation (all P values >.20). This study indicates that in patients with placenta previa, topical recombinant activated factor VII may diminish bleeding from the placenta site without initiation

  8. [Expression and activity determination of recombinant capsid protein VP2 gene of enterovirus type 71].

    Science.gov (United States)

    Huang, Xueyong; Liu, Guohua; Hu, Xiaoning; Du, Yanhua; Li, Xingle; Xu, Yuling; Chen, Haomin; Xu, Bianli

    2014-04-01

    To clone and express the recombinant capsid protein VP2 of enterovirus type 71 (EV71) and to identify the immune activity of expressed protein in order to build a basis for the investigation work of vaccine and diagnostic antigen. VP2 gene of EV71 was amplified by PCR, and then was cut by restriction enzyme and inserted into expression vector pMAL-c2X. The positive recombinants were transferred into E.coli TB1, the genetically engineered bacteria including pMAL-c2X-VP2 plasmids were induced by isopropyl thiogalactoside ( IPTG) , and the expression products were analyzed by SDS-PAGE and western blotting method. EV71 IgM antibody detection method by ELISA was set up, and the sensitivity and specificity of this method was assessed; 60 neutralizing antibody positive serum samples from hand foot and mouth disease (HFMD) patients were determined, of which 52 samples were positive and 8 samples were negative; a total of 88 acute phase serum samples of HFMD patients diagnosed in clinical were also detected. VP2 gene of 762 bp was obtained by PCR, the gene segment inserted into the recombinant vector was identified using restriction enzyme digestion. The recombinant vector could express a specific about 71 500 fusion protein in E.coli by SDS-PAGE. The purified recombinant protein of EV71-VP2 can react with the serum of HFMD patients to produce a specific band by western blotting. The sensitivity and specificity of ELISA was 87% and 83%, respectively. Of the 88 acute phase serum samples from children with HFMD, 48 samples (55%) were positive by the ELISA assay. VP2 gene of EV71 has been cloned and a prokaryotic high expression system for VP2 gene was successfully constructed in the present study. The recombination EV71-VP2 has well antigenicity, which could be useful for developing diagnose reagent or vaccine of EV71.

  9. Recombinant cold-adapted attenuated influenza A vaccines for use in children: reactogenicity and antigenic activity of cold-adapted recombinants and analysis of isolates from the vaccinees.

    OpenAIRE

    Alexandrova, G I; Polezhaev, F I; Budilovsky, G N; Garmashova, L M; Topuria, N A; Egorov, A Y; Romejko-Gurko, Y R; Koval, T A; Lisovskaya, K V; Klimov, A I

    1984-01-01

    Reactogenicity and antigenic activity of recombinants obtained by crossing cold-adapted donor of attenuation A/Leningrad/134/47/57 with wild-type influenza virus strains A/Leningrad/322/79(H1N1) and A/Bangkok/1/79(H3N2) were studied. The recombinants were areactogenic when administered as an intranasal spray to children aged 3 to 15, including those who lacked or had only low titers of pre-existing anti-hemagglutinin and anti-neuraminidase antibody in their blood. After two administrations of...

  10. Azadirachtin-induced apoptosis involves lysosomal membrane permeabilization and cathepsin L release in Spodoptera frugiperda Sf9 cells.

    Science.gov (United States)

    Wang, Zheng; Cheng, Xingan; Meng, Qianqian; Wang, Peidan; Shu, Benshui; Hu, Qiongbo; Hu, Meiying; Zhong, Guohua

    2015-07-01

    Azadirachtin as a kind of botanical insecticide has been widely used in pest control. We previously reported that azadirachtin could induce apoptosis of Spodoptera litura cultured cell line Sl-1, which involves in the up-regulation of P53 protein. However, the detailed mechanism of azadirachtin-induced apoptosis is not clearly understood in insect cultured cells. The aim of the present study was to address the involvement of lysosome and lysosomal protease in azadirachtin-induced apoptosis in Sf9 cells. The result confirmed that azadirachtin indeed inhibited proliferation and induced apoptosis. The lysosomes were divided into different types as time-dependent manner, which suggested that changes of lysosomes were necessarily physiological processes in azadirachtin-induced apoptosis in Sf9 cells. Interestingly, we noticed that azadirachtin could trigger lysosomal membrane permeabilization and cathepsin L releasing to cytosol. Z-FF-FMK (a cathepsin L inhibitor), but not CA-074me (a cathepsin B inhibitor), could effectively hinder the apoptosis induced by azadirachtin in Sf9 cells. Meanwhile, the activity of caspase-3 could also be inactivated by the inhibition of cathepsin L enzymatic activity induced by Z-FF-FMK. Taken together, our findings suggest that azadirachtin could induce apoptosis in Sf9 cells in a lysosomal pathway, and cathepsin L plays a pro-apoptosis role in this process through releasing to cytosol and activating caspase-3. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Insecticidal activity of two proteases against Spodoptera frugiperda larvae infected with recombinant baculoviruses

    Science.gov (United States)

    2010-01-01

    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

  12. Safety update on the use of recombinant activated factor VII in approved indications.

    Science.gov (United States)

    Neufeld, Ellis J; Négrier, Claude; Arkhammar, Per; Benchikh el Fegoun, Soraya; Simonsen, Mette Duelund; Rosholm, Anders; Seremetis, Stephanie

    2015-06-01

    This updated safety review summarises the large body of safety data available on the use of recombinant activated factor VII (rFVIIa) in approved indications: haemophilia with inhibitors, congenital factor VII (FVII) deficiency, acquired haemophilia and Glanzmann's thrombasthenia. Accumulated data up to 31 December 2013 from clinical trials as well as post-marketing data (registries, literature reports and spontaneous reports) were included. Overall, rFVIIa has shown a consistently favourable safety profile, with no unexpected safety concerns, in all approved indications. No confirmed cases of neutralising antibodies against rFVIIa have been reported in patients with congenital haemophilia, acquired haemophilia or Glanzmann's thrombasthenia. The favourable safety profile of rFVIIa can be attributed to the recombinant nature of rFVIIa and its localised mechanism of action at the site of vascular injury. Recombinant FVIIa activates factor X directly on the surface of activated platelets, which are present only at the site of injury, meaning that systemic activation of coagulation is avoided and the risk of thrombotic events (TEs) thus reduced. Nonetheless, close monitoring for signs and symptoms of TE is warranted in all patients treated with any pro-haemostatic agent, including rFVIIa, especially the elderly and any other patients with concomitant conditions and/or predisposing risk factors to thrombosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

  13. The control of neutrophil chemotaxis by inhibitors of cathepsin G and chymotrypsin.

    Science.gov (United States)

    Lomas, D A; Stone, S R; Llewellyn-Jones, C; Keogan, M T; Wang, Z M; Rubin, H; Carrell, R W; Stockley, R A

    1995-10-06

    Neutrophil chemotaxis plays an important role in the inflammatory response and when excessive or persistent may augment tissue damage. The effects of inhibitors indicated the involvement of one or more serine proteinases in human neutrophil migration and shape change in response to a chemoattractant. Monospecific antibodies, chloromethylketone inhibitors, and reactive-site mutants of alpha 1-antitrypsin and alpha 1-antichymotrypsin were used to probe the specificity of the proteinases involved in chemotaxis. Antibodies specific for cathepsin G inhibited chemotaxis. Moreover, rapid inhibitors of cathepsin G and alpha-chymotrypsin suppressed neutrophil chemotaxis to the chemoattractants N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP) and zymosan-activated serum in multiple blind well assays and to fMLP in migration assays under agarose. The concentrations of antichymotrypsin mutants that reduced chemotaxis by 50% would inactivate free cathepsin G with a half-life of 1.5-3 s, whereas the concentrations of chloromethylketones required to produce a similar inhibition of chemotaxis would inactivate cathepsin G with a half-life of 345 s. These data suggest different modes of action for these two classes of inhibitors. Indeed the chloromethylketone inhibitors of cathepsin G (Z-Gly-Leu-Phe-CMK) and to a lesser extent of chymotrypsin (Cbz-Gly-Gly-Phe-CMK) mediated their effect by preventing a shape change in the purified neutrophils exposed to fMLP. Antichymotrypsin did not affect shape change in response to fMLP even at concentrations that were able to reduce neutrophil chemotaxis by 50%. These results support the involvement of cell surface proteinases in the control of cell migration and show that antichymotrypsin and chloromethylketones have differing modes of action. This opens the possibility for the rational design of anti-inflammatory agents targeted at neutrophil membrane enzymes.

  14. Immunoadjuvant activities of a recombinant chicken IL-12 in chickens vaccinated with Newcastle disease virus recombinant HN protein.

    Science.gov (United States)

    Su, Bor Sheu; Yin, Hsien Sheng; Chiu, Hua Hsien; Hung, Li Hsiang; Huang, Ji Ping; Shien, Jui Hung; Lee, Long Huw

    2011-08-05

    Recombinant fowlpox virus (rFPV/HN) expressing Newcastle disease virus (NDV) HN gene and rFPV/HN/chIL-12 co-expressing chicken IL-12 (chIL-12) and HN (rHN/chIL-12) genes have been characterized. rHN/chIL-12 or rchIL-12, expressed by our previous construct rFPV/chIL-12, co-administered with rHN was assessed for adjuvant activities of chIL-12. Chickens were vaccinated with various amounts of rHN/chIL-12 mixed with mineral oil (MO), intramuscularly. Levels of hemagglutination-inhibition (HI) antibody production depended on the concentration of the injected rHN or rHN/chIL-12. The lower HI antibody titers were obtained in chicken groups rHN/chIL-12/7-rHN/chIL-12/9, receiving 60ng rHN/8ng chIL-12 with MO, 30ng rHN/4ng chIL-12 with MO or 15ng rHN/2ng chIL-12 with MO, respectively, compared to those in chicken groups rHN/7-rHN/9, receiving rHN with MO alone. However, chickens in group rHN/chIL-12/7 or rHN/chIL-12/8 and rHN with MO alone showed the same effective protection. Chicken group rHN/chIL-12/9 was even more protective than that in group rHN/9. When rchIL-12 was co-injected with 15ng rHN plus MO, chickens produced low levels of HI antibody titers; while higher levels of IFN-γ production and an effective protection rate (83%) were obtained. On the other hand, low levels of IFN-γ production and low protection response (50%) were obtained in chickens injected with rHN with MO alone. Taken together, when the concentration of rHN decreased to certain levels, rchIL-12 reduced HI antibody production. The increase in the induction of IFN-γ production might suggest the enhancement of the cell-mediated immunity which conferred the protection from the NDV challenge. Copyright © 2011 Elsevier B.V. All rights reserved.

  15. Soluble multimer of recombinant endostatin expressed in E. coli has anti-angiogenesis activity

    International Nuclear Information System (INIS)

    Wei Dongmei; Gao Yan; Cao Xiangrong; Zhu Nianchun; Liang Jianfu; Xie Weiping; Zhen Mingying; Zhu Minsheng

    2006-01-01

    The bioactivity, refolding, and multimer formation of endostatin, particularly of recombinant endostatin produced from bacteria, are proved challenging for clinical application. In order to determine the biological activity of recombinant endostatin multimer, first, we expressed endostatin in Escherichia coli and purified it with ion-exchange chromatography. The purified active protein could elicit multimer formation spontaneously, but still has comparable activity. Aim to determine the anti-angiogenic activity of multimer endostatin, by use of RP-HPLC, we then successfully separated endostatin monomer and multimer for subjecting to anti-angiogenesis assay. The results from CAM (chorioallantoic membrane) inhibition assay showed that both monomer and multimer suppressed CAM vascularization significantly. At the dosage of 0.8 μg, inhibition rates of multimeric and monomeric proteins were about 58% and 38%, respectively. Multimeric endostatin exerted a higher activity than monomeric endostatin (p 0.05), although both of them show a high inhibition effect in contrast to control. The results from HUVEC proliferation assay also showed similar effects at dosages of 0.6 and 1.6 μg/ml, multimer exerted a higher activity on inhibition of HUVEC proliferation comparing with monomer (p < 0.05). In conclusion, our results suggest that endostatin multimer has a comparable or higher bioactivity and multimerization will not affect its bioactivity, implying that endostatin activity is insensitive to structure conformation contributed by disulfide bonds

  16. An Ectosteric Inhibitor of Cathepsin K Inhibits Bone Resorption in Ovariectomized Mice

    DEFF Research Database (Denmark)

    Panwar, Preety; Xue, Liming; Søe, Kent

    2017-01-01

    The potent cathepsin K (CatK) inhibitor, Tanshinone IIA sulfonic sodium (T06), was tested for its in vitro and in vivo antiresorptive activities. T06 binds in an ectosteric site of CatK remote from its active site and selectively inhibits collagen degradation with an IC50 value of 2.7±0.2μM (CatK...

  17. Construction and expression of a recombinant antibody-targeted plasminogen activator

    International Nuclear Information System (INIS)

    Schnee, J.M.; Runge, M.S.; Matsueda, G.A.; Hudson, N.W.; Seidman, J.G.; Haber, E.; Quertermous, T.

    1987-01-01

    Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin β chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent that t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, the authors have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the γ2b constant region and the catalytic β chain of t-PA. This construct was transfected into heavy chain loss variant cells derived form the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots and radioimmunoassay. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. IN a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, they have produced a hybrid protein capable of high affinity fibrin binding and plasminogen activation

  18. Plasma levels of cathepsins L, K, and V and risks of abdominal aortic aneurysms

    DEFF Research Database (Denmark)

    Lv, Bing-Jie; Lindholt, Jes S; Wang, Jing

    2013-01-01

    Cathepsin L (CatL), cathepsin K (CatK), and cathepsin V (CatV) are potent elastases implicated in human arterial wall remodeling. Whether plasma levels of these cathepsins are altered in patients with abdominal aortic aneurysms (AAAs) remains unknown....

  19. Catalytic activity of metallic nanoisland coatings. The influence of size effects on the recombination properties

    International Nuclear Information System (INIS)

    Tomilina, O A; Berzhansky, V N; Shaposhnikov, A N; Tomilin, S V

    2016-01-01

    The results of investigations of the quantum-size effects influence on selective properties of heterogeneous nanocatalysts are presents. As etalon exothermic reaction was used the reaction of atomic hydrogen recombination. The nanostructured Pd and Pt films on Teflon substrate were used as a samples of heterogeneous nanocatalysts. It was shown that for nanoparticles with various sizes the catalytic activity has the periodic dependence. It has been found that for certain sizes of nanoparticles their catalytic activity is less than that of Teflon substrate. (paper)

  20. A cardinal role for cathepsin d in co-ordinating the host-mediated apoptosis of macrophages and killing of pneumococci.

    Directory of Open Access Journals (Sweden)

    Martin A Bewley

    2011-01-01

    Full Text Available The bactericidal function of macrophages against pneumococci is enhanced by their apoptotic demise, which is controlled by the anti-apoptotic protein Mcl-1. Here, we show that lysosomal membrane permeabilization (LMP and cytosolic translocation of activated cathepsin D occur prior to activation of a mitochondrial pathway of macrophage apoptosis. Pharmacological inhibition or knockout of cathepsin D during pneumococcal infection blocked macrophage apoptosis. As a result of cathepsin D activation, Mcl-1 interacted with its ubiquitin ligase Mule and expression declined. Inhibition of cathepsin D had no effect on early bacterial killing but inhibited the late phase of apoptosis-associated killing of pneumococci in vitro. Mice bearing a cathepsin D(-/- hematopoietic system demonstrated reduced macrophage apoptosis in vivo, with decreased clearance of pneumococci and enhanced recruitment of neutrophils to control pulmonary infection. These findings establish an unexpected role for a cathepsin D-mediated lysosomal pathway of apoptosis in pulmonary host defense and underscore the importance of apoptosis-associated microbial killing to macrophage function.

  1. Cre-dependent DNA recombination activates a STING-dependent innate immune response

    Science.gov (United States)

    Pépin, Geneviève; Ferrand, Jonathan; Höning, Klara; Jayasekara, W. Samantha N.; Cain, Jason E.; Behlke, Mark A.; Gough, Daniel J.; G. Williams, Bryan R.; Hornung, Veit

    2016-01-01

    Abstract Gene-recombinase technologies, such as Cre/loxP-mediated DNA recombination, are important tools in the study of gene function, but have potential side effects due to damaging activity on DNA. Here we show that DNA recombination by Cre instigates a robust antiviral response in mammalian cells, independent of legitimate loxP recombination. This is due to the recruitment of the cytosolic DNA sensor STING, concurrent with Cre-dependent DNA damage and the accumulation of cytoplasmic DNA. Importantly, we establish a direct interplay between this antiviral response and cell–cell interactions, indicating that low cell densities in vitro could be useful to help mitigate these effects of Cre. Taking into account the wide range of interferon stimulated genes that may be induced by the STING pathway, these results have broad implications in fields such as immunology, cancer biology, metabolism and stem cell research. Further, this study sets a precedent in the field of gene-engineering, possibly applicable to other enzymatic-based genome editing technologies. PMID:27166376

  2. Refolding Techniques for Recovering Biologically Active Recombinant Proteins from Inclusion Bodies

    Directory of Open Access Journals (Sweden)

    Hiroshi Yamaguchi

    2014-02-01

    Full Text Available Biologically active proteins are useful for studying the biological functions of genes and for the development of therapeutic drugs and biomaterials in a biotechnology industry. Overexpression of recombinant proteins in bacteria, such as Escherichia coli, often results in the formation of inclusion bodies, which are protein aggregates with non-native conformations. As inclusion bodies contain relatively pure and intact proteins, protein refolding is an important process to obtain active recombinant proteins from inclusion bodies. However, conventional refolding methods, such as dialysis and dilution, are time consuming and, often, recovered yields of active proteins are low, and a trial-and-error process is required to achieve success. Recently, several approaches have been reported to refold these aggregated proteins into an active form. The strategies largely aim at reducing protein aggregation during the refolding procedure. This review focuses on protein refolding techniques using chemical additives and laminar flow in microfluidic chips for the efficient recovery of active proteins from inclusion bodies.

  3. Role of Cathepsin S in Periodontal Inflammation and Infection

    Directory of Open Access Journals (Sweden)

    S. Memmert

    2017-01-01

    Full Text Available Cathepsin S is a cysteine protease and regulator of autophagy with possible involvement in periodontitis. The objective of this study was to investigate whether cathepsin S is involved in the pathogenesis of periodontal diseases. Human periodontal fibroblasts were cultured under inflammatory and infectious conditions elicited by interleukin-1β and Fusobacterium nucleatum, respectively. An array-based approach was used to analyze differential expression of autophagy-associated genes. Cathepsin S was upregulated most strongly and thus further studied in vitro at gene and protein levels. In vivo, gingival tissue biopsies from rats with ligature-induced periodontitis and from periodontitis patients were also analyzed at transcriptional and protein levels. Multiple gene expression changes due to interleukin-1β and F. nucleatum were observed in vitro. Both stimulants caused a significant cathepsin S upregulation. A significantly elevated cathepsin S expression in gingival biopsies from rats with experimental periodontitis was found in vivo, as compared to that from control. Gingival biopsies from periodontitis patients showed a significantly higher cathepsin S expression than those from healthy gingiva. Our findings provide original evidence that cathepsin S is increased in periodontal cells and tissues under inflammatory and infectious conditions, suggesting a critical role of this autophagy-associated molecule in the pathogenesis of periodontitis.

  4. Top3 processes recombination intermediates and modulates checkpoint activity after DNA damage

    DEFF Research Database (Denmark)

    Mankouri, Hocine W; Hickson, Ian D

    2006-01-01

    Mutation of TOP3 in Saccharomyces cerevisiae causes poor growth, hyperrecombination, and a failure to fully activate DNA damage checkpoints in S phase. Here, we report that overexpression of a dominant-negative allele of TOP3, TOP3(Y356F), which lacks the catalytic (decatenation) activity of Top3......, the catalytic activity of Top3 is not required for DNA damage checkpoint activation, but it is required for normal S-phase progression after DNA damage. We also present evidence that the checkpoint-mediated cell cycle delay and persistence of X-shaped DNA molecules resulting from overexpression of TOP3(Y356F......) are downstream of Rad51 function. We propose that Top3 functions in S phase to both process homologous recombination intermediates and modulate checkpoint activity....

  5. Biological activity analysis of native and recombinant streptokinase using clot lysis and chromogenic substrate assay.

    Science.gov (United States)

    Mahboubi, Arash; Sadjady, Seyyed Kazem; Mirzaei Saleh Abadi, Mohammad; Azadi, Saeed; Solaimanian, Roya

    2012-01-01

    DETERMINATION OF STREPTOKINASE ACTIVITY IS USUALLY ACCOMPLISHED THROUGH TWO ASSAY METHODS: a) Clot lysis, b) Chromogenic substrate assay. In this study the biological activity of two streptokinase products, namely Streptase®, which is a native product and Heberkinasa®, which is a recombinant product, was determined against the third international reference standard using the two forementioned assay methods. The results indicated that whilst the activity of Streptase® was found to be 101 ± 4% and 97 ± 5% of the label claim with Clot lysis and Chromogenic substrate assay respectively, for Heberkinasa® the potency values obtained were 42 ± 5% and 92.5 ± 2% of the label claim respectively. To shed some light on the reason for this finding, the n-terminal sequence of the streptokinase molecules present in the two products was determined. The results showed slight differences in the amino acid sequence of the recombinant product in comparison to the native one at the amino terminus. This finding supports those of other workers who found that n-terminal sequence of the streptokinase molecule can have significant effect on the activity of this protein.

  6. Active site electrostatics protect genome integrity by blocking abortive hydrolysis during DNA recombination

    Science.gov (United States)

    Ma, Chien-Hui; Rowley, Paul A; Macieszak, Anna; Guga, Piotr; Jayaram, Makkuni

    2009-01-01

    Water, acting as a rogue nucleophile, can disrupt transesterification steps of important phosphoryl transfer reactions in DNA and RNA. We have unveiled this risk, and identified safeguards instituted against it, during strand cleavage and joining by the tyrosine site-specific recombinase Flp. Strand joining is threatened by a latent Flp endonuclease activity (type I) towards the 3′-phosphotyrosyl intermediate resulting from strand cleavage. This risk is not alleviated by phosphate electrostatics; neutralizing the negative charge on the scissile phosphate through methylphosphonate (MeP) substitution does not stimulate type I endonuclease. Rather, protection derives from the architecture of the recombination synapse and conformational dynamics within it. Strand cleavage is protected against water by active site electrostatics. Replacement of the catalytic Arg-308 of Flp by alanine, along with MeP substitution, elicits a second Flp endonuclease activity (type II) that directly targets the scissile phosphodiester bond in DNA. MeP substitution, combined with appropriate active site mutations, will be useful in revealing anti-hydrolytic mechanisms engendered by systems that mediate DNA relaxation, DNA transposition, site-specific recombination, telomere resolution, RNA splicing and retrohoming of mobile introns. PMID:19440204

  7. The Meiotic Recombination Activator PRDM9 Trimethylates Both H3K36 and H3K4 at Recombination Hotspots In Vivo.

    Science.gov (United States)

    Powers, Natalie R; Parvanov, Emil D; Baker, Christopher L; Walker, Michael; Petkov, Petko M; Paigen, Kenneth

    2016-06-01

    In many mammals, including humans and mice, the zinc finger histone methyltransferase PRDM9 performs the first step in meiotic recombination by specifying the locations of hotspots, the sites of genetic recombination. PRDM9 binds to DNA at hotspots through its zinc finger domain and activates recombination by trimethylating histone H3K4 on adjacent nucleosomes through its PR/SET domain. Recently, the isolated PR/SET domain of PRDM9 was shown capable of also trimethylating H3K36 in vitro, raising the question of whether this reaction occurs in vivo during meiosis, and if so, what its function might be. Here, we show that full-length PRDM9 does trimethylate H3K36 in vivo in mouse spermatocytes. Levels of H3K4me3 and H3K36me3 are highly correlated at hotspots, but mutually exclusive elsewhere. In vitro, we find that although PRDM9 trimethylates H3K36 much more slowly than it does H3K4, PRDM9 is capable of placing both marks on the same histone molecules. In accord with these results, we also show that PRDM9 can trimethylate both K4 and K36 on the same nucleosomes in vivo, but the ratio of K4me3/K36me3 is much higher for the pair of nucleosomes adjacent to the PRDM9 binding site compared to the next pair further away. Importantly, H3K4me3/H3K36me3-double-positive nucleosomes occur only in regions of recombination: hotspots and the pseudoautosomal (PAR) region of the sex chromosomes. These double-positive nucleosomes are dramatically reduced when PRDM9 is absent, showing that this signature is PRDM9-dependent at hotspots; the residual double-positive nucleosomes most likely come from the PRDM9-independent PAR. These results, together with the fact that PRDM9 is the only known mammalian histone methyltransferase with both H3K4 and H3K36 trimethylation activity, suggest that trimethylation of H3K36 plays an important role in the recombination process. Given the known requirement of H3K36me3 for double strand break repair by homologous recombination in somatic cells, we

  8. Does intravenous administration of recombinant tissue plasminogen activator for ischemic stroke can cause inferior myocardial infarction?

    Directory of Open Access Journals (Sweden)

    Mostafa Almasi

    2016-06-01

    Full Text Available Recombinant tissue plasminogen activator (rTPA is one of the main portions of acute ischemic stroke management, but unfortunately has some complications. Myocardial infarction (MI is a hazardous complication of administration of intravenous rTPA that has been reported recently. A 78-year-old lady was admitted for elective coronary artery bypass graft surgery. On the second day of admission, she developed acute left hemiparesis and intravenous rTPA was administered within 120 minutes. Three hours later, she has had chest pain. Rescue percutaneous coronary intervention was performed on right coronary artery due to diagnosis of inferior MI, and the symptoms were resolved.

  9. Cathepsin C and plasma glutamate carboxypeptidase secreted from Fischer rat thyroid cells liberate thyroxin from the N-terminus of thyroglobulin.

    Science.gov (United States)

    Suban, Dejan; Zajc, Tajana; Renko, Miha; Turk, Boris; Turk, Vito; Dolenc, Iztok

    2012-03-01

    The release of a thyroid hormone from thyroglobulin is controlled by a complex regulatory system. We focused on the extracellular action of two lysosomal enzymes, cathepsin C (catC, dipeptidyl peptidase I) and PGCP (lysosomal dipeptidase), on thyroglobulin, and their ability to liberate the hormone thyroxin. Cathepsin C, an exopeptidase, removes dipeptides from the N-terminus of substrates, and PGCP hydrolyses dipeptides to amino acids. In vitro experiments proved that cathepsin C removes up to 12 amino acids from the N-terminus of porcine thyroglobulin, including a dipeptide with thyroxin on position 5. The newly formed N-terminus, Arg-Pro-, was not hydrolysed further by cathepsin C. Cell culture experiments with FRTL-5 cell line showed localization of cathepsin C and PGCP and their secretion into the medium. Secretion of the active cathepsin C from FRTL-5 cells is stimulated by TSH, insulin, and/or somatostatin. The released enzymes liberate thyroxin from porcine thyroglobulin added to media. The hormone liberation can be reduced by synthetic inhibitors of cysteine proteinases and metalloproteinases. Additionally, we show that TSH, insulin, and/or somatostatin induce up-regulation of N-acetylglucosaminyltransferase 1, the enzyme responsible for the initiation of biosynthesis of hybrid and complex N-glycosylation of proteins. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  10. Production and characterization of guinea pig recombinant gamma interferon and its effect on macrophage activation.

    Science.gov (United States)

    Jeevan, A; McFarland, C T; Yoshimura, T; Skwor, T; Cho, H; Lasco, T; McMurray, D N

    2006-01-01

    Gamma interferon (IFN-gamma) plays a critical role in the protective immune responses against mycobacteria. We previously cloned a cDNA coding for guinea pig IFN-gamma (gpIFN-gamma) and reported that BCG vaccination induced a significant increase in the IFN-gamma mRNA expression in guinea pig cells in response to living mycobacteria and that the virulent H37Rv strain of Mycobacterium tuberculosis stimulated less IFN-gamma mRNA than did the attenuated H37Ra strain. In this study, we successfully expressed and characterized recombinant gpIFN-gamma with a histidine tag at the N terminus (His-tagged rgpIFN-gamma) in Escherichia coli. rgpIFN-gamma was identified as an 18-kDa band in the insoluble fraction; therefore, the protein was purified under denaturing conditions and renatured. N-terminal amino acid sequencing of the recombinant protein yielded the sequence corresponding to the N terminus of His-tagged gpIFN-gamma. The recombinant protein upregulated major histocompatibility complex class II expression in peritoneal macrophages. The antiviral activity of rgpIFN-gamma was demonstrated with a guinea pig fibroblast cell line (104C1) infected with encephalomyocarditis virus. Interestingly, peritoneal macrophages treated with rgpIFN-gamma did not produce any nitric oxide but did produce hydrogen peroxide and suppressed the intracellular growth of mycobacteria. Furthermore, rgpIFN-gamma induced morphological alterations in cultured macrophages. Thus, biologically active rgpIFN-gamma has been successfully produced and characterized in our laboratory. The study of rgpIFN-gamma will further increase our understanding of the cellular and molecular responses induced by BCG vaccination in the guinea pig model of pulmonary tuberculosis.

  11. Technetium-99m-labeled recombinant tissue plasminogen activator for the imaging of emboli in vivo

    Energy Technology Data Exchange (ETDEWEB)

    Takahashi, Akihiro; Itoh, Kazuo; Tsukamoto, Eriko; Furudate, Masayori; Kamiyama, Hiroyasu; Abe, Hiroshi [Hokkaido Univ., Sapporo (Japan). School of Medicine

    1993-07-01

    Tissue-type plasminogen activator (t-PA) effectively lyses activate thrombus by direct action. Recombinant t-PA (rt-PA) was labeled with technetium-99m ([sup 99m]Tc) to investigate the in vivo binding to fibrin clots in a feline cerebral embolism model created by insertion of an artificial fibrin clot within the carotid artery. [sup 99m]Tc-rt-PA administered intravenously provided clearer imaging of clots after priming with cold rt-PA, with uptake peaking 5-10 minutes after the injection. [sup 99m]Tc-labeled human serum albumin was not retained at clot sites. Systemically administered [sup 99m]Tc-rt-PA binds to fibrin clots within carotid arteries in our feline model. Our results suggest that the interaction of intrinsic plasminogen activator inhibitors with extrinsically administered rt-PA may regulate the demonstration of a clot, although the precise mechanism is unclear. (author).

  12. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Science.gov (United States)

    Dolinska, Monika B; Kovaleva, Elena; Backlund, Peter; Wingfield, Paul T; Brooks, Brian P; Sergeev, Yuri V

    2014-01-01

    Tyrosinase (TYR) catalyzes the rate-limiting, first step in melanin production and its gene (TYR) is mutated in many cases of oculocutaneous albinism (OCA1), an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes. The intra-melanosomal domain of human tyrosinase (residues 19-469) and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure. The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  13. Albinism-causing mutations in recombinant human tyrosinase alter intrinsic enzymatic activity.

    Directory of Open Access Journals (Sweden)

    Monika B Dolinska

    Full Text Available Tyrosinase (TYR catalyzes the rate-limiting, first step in melanin production and its gene (TYR is mutated in many cases of oculocutaneous albinism (OCA1, an autosomal recessive cause of childhood blindness. Patients with reduced TYR activity are classified as OCA1B; some OCA1B mutations are temperature-sensitive. Therapeutic research for OCA1 has been hampered, in part, by the absence of purified, active, recombinant wild-type and mutant human enzymes.The intra-melanosomal domain of human tyrosinase (residues 19-469 and two OCA1B related temperature-sensitive mutants, R422Q and R422W were expressed in insect cells and produced in T. ni larvae. The short trans-membrane fragment was deleted to avoid potential protein insolubility, while preserving all other functional features of the enzymes. Purified tyrosinase was obtained with a yield of >1 mg per 10 g of larval biomass. The protein was a monomeric glycoenzyme with maximum enzyme activity at 37°C and neutral pH. The two purified mutants when compared to the wild-type protein were less active and temperature sensitive. These differences are associated with conformational perturbations in secondary structure.The intramelanosomal domains of recombinant wild-type and mutant human tyrosinases are soluble monomeric glycoproteins with activities which mirror their in vivo function. This advance allows for the structure - function analyses of different mutant TYR proteins and correlation with their corresponding human phenotypes; it also provides an important tool to discover drugs that may improve tyrosinase activity and treat OCA1.

  14. 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

    2011-09-01

    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.

  15. Recombinant-activated factor VII in patients with uncontrolled bleeding: A retrospective observational analysis

    Directory of Open Access Journals (Sweden)

    Said D Abuhasna

    2012-01-01

    Full Text Available Background: Factor VIIa (recombinant has an off-label use to control life-threatening bleeding that is refractory to other measures and was shown to decrease transfusion requirements. Objective: The primary objective of this study was to assess the safety and effectiveness of factor VIIa (recombinant on blood transfusion requirements and coagulation parameters when used in patients whose bleeding was uncorrected by other means. The pharmacoeconomic impact for any discrepancy from our protocol was evaluated. Secondary outcomes included 4-hour and 28-day mortality, as well as safety of this agent in terms of thromboembolic complications. Materials and Methods: We retrospectively evaluated patients who received recombinant-activated factor VII (rFVIIa for uncontrolled bleeding from June 2008 to April 2011. The medical records of 33 patients were evaluated. Coagulation parameters and blood products were determined 24 hours before and 24 hours after administration of rFVIIa, and the results compared. Patients were also screened for any thromboembolic complications. Results: Administration of rFVIIa reduced blood transfusion requirements and improved coagulation parameters significantly (P<0.05. No thromboembolic complications were reported. Most of the dosing was consistent with those recommended in our institutional protocol, with discrepancies resulting in an average cost of $56 058. Moreover, pH was reported in only 67% of patients. All patients treated with rFVIIa survived up to 4 hours after receiving this agent, while the 28-day mortality was 24% (8/33. Conclusion: The use of rFVIIa appears to be safe and effective in promoting hemostasis, as evident from reducing transfusion requirements and improving the coagulation variables.

  16. Construction and Antiapoptosis Activities of Recombinant Adenoviral Expression Vector Carrying EBV Latent Membrane Protein 2A

    Directory of Open Access Journals (Sweden)

    Xishuang Liu

    2011-01-01

    Full Text Available To evaluate the possible effects of LMP2A (EBV latent membrane protein 2A on human gastric cancer cell line SGC-7901, LMP2A coding gene was subcloned into shuttle plasmid pAdTrackCMV to form transfer plasmid pAdTrackCMV-2A, which was linearized with PmeI and cotransformed into E.coli BJ5183 with adenovirus genomic plasmid of pAdeasy-1. The identified recombinant adenovirus plasmid DNA was digested with PacI and transfected into 293 cells to package recombinant adenovirus particles named vAd-2A. Then the expression and antiapoptosis activities of LMP2A on SGC-7901 infected with vAd-2A were analyzed. The vAd-2A was successfully constructed and identified by PCR, restriction digestion, and sequencing. LMP2A expression in SGC was identified by strong green fluorescence expression with fluorescence microscopic photograph and Southern blotting. The growth of LMP2A expressing SGC cells was apparently improved. Both cyclin E expression and S phase ratio in LMP2A expressing SGC cells were upregulated by cell cycle analysis and confocal microscopic analysis respectively. The replication-deficient recombinant adenovirus vector can express LMP2A antigen in SGC cells and inhibit their apoptosis. The results indicate that LMP2A might play an important role in pathogenesis of EBV-associated gastric cancer (EBVaGC. This study establishes a foundation for further study on EBVaGC and its gene therapy.

  17. Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

    Science.gov (United States)

    Gerashchenko, O L; Zhuravel, E V; Skachkova, O V; Khranovska, N N; Filonenko, V V; Pogrebnoy, P V; Soldatkina, M A

    2013-06-01

    The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.

  18. Evaluation of Aryoseven Safety (Recombinant Activated Factor VII) in Patients with Bleeding Disorders (An Observational Post-Marketing Surveillance Study).

    Science.gov (United States)

    Toogeh, Gholamreza; Abolghasemi, Hassan; Eshghi, Peyman; Managhchi, Mohammadreza; Shaverdi-Niasari, Mohammadreza; Karimi, Katayoon; Roostaei, Samin; Emran, Neda; Abdollahi, Alireza

    2016-01-01

    Recombinant activated factor VII induces hemostasis in patients with coagulopathy disorders. AryoSeven™ as a safe Iranian Recombinant activated factor VII has been available on our market. This study was performed to establish the safety of AryoSeven on patients with coagulopathy disorder. This single-center, descriptive, cross sectional study was carried out in Thrombus and Homeostasis Research Center ValiAsr Hospital during 2013-2014. Fifty one patients with bleeding disorders who received at least one dose of Aryoseven were enrolled. Patients' demographic data and adverse effect of drug and reaction related to Aryoseven or previous usage of Recombinant activated FVII were recorded in questionnaires. Finally data were analyzed to compare side effects of Aryoseven and other Recombinant activated FVII brands. Aryoseven was prescribed for 51 Patients. Of all participants with mean age 57.18+21.38 yr, 31 cases were male and 26 subjects had past history of recombinant activated FVII usage. Glanzman was the most frequent disorder followed by congenital FVII deficiency, hemophilia with inhibitors, factor 5 deficiency, acquired hemophilia, hemophilia A with inhibitor, and hemophilia A or B with inhibitor. The majority of bleeding episodes had occurred in joints. Three patients (5.9%) complained about adverse effects of Aryoseven vs. 11.5 % about adverse effects of other brands. However this difference was not significant, statistically. Based on monitor patients closely for any adverse events, we concluded that Aryoseven administration under careful weighing of benefit versus potential harm may comparable with other counterpart drugs.

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

    Directory of Open Access Journals (Sweden)

    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. Chromate-reducing activity of Hansenula polymorpha recombinant cells over-producing flavocytochrome b₂.

    Science.gov (United States)

    Smutok, Oleh; Broda, Daniel; Smutok, Halyna; Dmytruk, Kostyantyn; Gonchar, Mykhailo

    2011-04-01

    In spite of the great interest to studies of the biological roles of chromium, as well as the toxic influence of Cr(VI)-species on living organisms, the molecular mechanisms of chromate bioremediation remain vague. A reductive pathway resulting in formation of less toxic Cr(III)-species is suggested to be the most important among possible mechanisms for chromate biodetoxification. The yeast l-lactate:cytochrome c-oxidoreductase (flavocytochrome b(2), FC b(2)) has absolute specificity for l-lactate, yet is non-selective with respect to its electron acceptor. These properties allow us to consider the enzyme as a potential candidate for chromate reduction by living cells in the presence of l-lactate. A recombinant strain of thermotolerant, methylotrophic yeast Hansenula polymorpha with sixfold increased FC b(2) enzyme activity (up to 3μmolmin(-1)mg(-1) protein in cell-free extract) compared to the parental strain was used for approval our suggestion. The recombinant cells, stored in dried state, as well as living yeast cells were tested for chromate-reducing activity in vitro in the presence of l-lactate (as an electron donor for chromate reduction) and different low molecular weight, redox-active mediators facilitating electron transfer from the reduced form of the enzyme to chromate (as a final electron acceptor): dichlorophenolindophenol (DCPIP), Methylene blue, Meldola blue, and Nile blue. It was shown that the highest chromate-reducing activity of the cells was achieved in the presence of DCPIP. The ability of chromate to catch electrons from the reduced flavocytochrome b(2) was confirmed using purified enzyme immobilized on the surface of a platinum electrode. The increasing concentration of Cr(VI) resulted in a decrease of enzyme-mediated current generated on the electrode during l-lactate oxidation. The shift and drop in amplitude of the peak in the cyclic voltammogram are indicative of Cr(VI)-dependent competition between reaction of chromate with reduced FC

  1. Perlecan domain 1 recombinant proteoglycan augments BMP-2 activity and osteogenesis

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    DeCarlo Arthur A

    2012-09-01

    Full Text Available Abstract Background Many growth factors, such as bone morphogenetic protein (BMP-2, have been shown to interact with polymers of sulfated disacharrides known as heparan sulfate (HS glycosaminoglycans (GAGs, which are found on matrix and cell-surface proteoglycans throughout the body. HS GAGs, and some more highly sulfated forms of chondroitin sulfate (CS, regulate cell function by serving as co-factors, or co-receptors, in GF interactions with their receptors, and HS or CS GAGs have been shown to be necessary for inducing signaling and GF activity, even in the osteogenic lineage. Unlike recombinant proteins, however, HS and CS GAGs are quite heterogenous due, in large part, to post-translational addition, then removal, of sulfate groups to various positions along the GAG polymer. We have, therefore, investigated whether it would be feasible to deliver a DNA pro-drug to generate a soluble HS/CS proteoglycan in situ that would augment the activity of growth-factors, including BMP-2, in vivo. Results Utilizing a purified recombinant human perlecan domain 1 (rhPln.D1 expressed from HEK 293 cells with HS and CS GAGs, tight binding and dose-enhancement of rhBMP-2 activity was demonstrated in vitro. In vitro, the expressed rhPln.D1 was characterized by modification with sulfated HS and CS GAGs. Dose-enhancement of rhBMP-2 by a pln.D1 expression plasmid delivered together as a lyophilized single-phase on a particulate tricalcium phosphate scaffold for 6 or more weeks generated up to 9 fold more bone volume de novo on the maxillary ridge in a rat model than in control sites without the pln.D1 plasmid. Using a significantly lower BMP-2 dose, this combination provided more than 5 times as much maxillary ridge augmentation and greater density than rhBMP-2 delivered on a collagen sponge (InFuse™. Conclusions A recombinant HS/CS PG interacted strongly and functionally with BMP-2 in binding and cell-based assays, and, in vivo, the pln.247 expression plasmid

  2. White shrimp (Litopenaeus vannamei) recombinant lysozyme has antibacterial activity against Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae.

    Science.gov (United States)

    de-la-Re-Vega, Enrique; García-Galaz, Alfonso; Díaz-Cinco, Martha E; Sotelo-Mundo, Rogerio R

    2006-03-01

    C-type lysozyme has been described as an antibacterial component of the shrimp innate defence system. We determined quantitatively the antibacterial activity of white shrimp (Litopenaeus vannamei) recombinant lysozyme against three Gram negative bacteria: Vibrio alginolyticus, Vibrio parahemolyticus and Vibrio cholerae, using a turbidimetric assay with live bacteria and differential bacterial viable count after interaction with the protein. In conclusion, the antibacterial activity of recombinant shrimp lysozyme against Vibrio sp. is at least equal to the values against the Gram positive M. luteus and more active against the shrimp pathogens V. alginolyticus and V. parahemolyticus.

  3. Residual endotoxin contaminations in recombinant proteins are sufficient to activate human CD1c+ dendritic cells.

    Directory of Open Access Journals (Sweden)

    Harald Schwarz

    Full Text Available Many commercially available recombinant proteins are produced in Escherichia coli, and most suppliers guarantee contamination levels of less than 1 endotoxin unit (EU. When we analysed commercially available proteins for their endotoxin content, we found contamination levels in the same range as generally stated in the data sheets, but also some that were higher. To analyse whether these low levels of contamination have an effect on immune cells, we stimulated the monocytic cell line THP-1, primary human monocytes, in vitro differentiated human monocyte-derived dendritic cells, and primary human CD1c+ dendritic cells (DCs with very low concentrations of lipopolysaccharide (LPS; ranging from 0.002-2 ng/ml. We show that CD1c+ DCs especially can be activated by minimal amounts of LPS, equivalent to the levels of endotoxin contamination we detected in some commercially available proteins. Notably, the enhanced endotoxin sensitivity of CD1c+ DCs was closely correlated with high CD14 expression levels observed in CD1c+ DCs that had been maintained in cell culture medium for 24 hours. When working with cells that are particularly sensitive to LPS, even low endotoxin contamination may generate erroneous data. We therefore recommend that recombinant proteins be thoroughly screened for endotoxin contamination using the limulus amebocyte lysate test, fluorescence-based assays, or a luciferase based NF-κB reporter assay involving highly LPS-sensitive cells overexpressing TLR4, MD-2 and CD14.

  4. Antibacterial activity and phospholipid recognition of the recombinant defensin J1-1 from Capsicum genus.

    Science.gov (United States)

    Guillén-Chable, Francisco; Arenas-Sosa, Iván; Islas-Flores, Ignacio; Corzo, Gerardo; Martinez-Liu, Cynthia; Estrada, Georgina

    2017-08-01

    The gene of the four disulfide-bridged defensin J1-1 from Capsicum was cloned into the expression vector pQE30 containing a 6His-tag as fusion protein. This construct was transfected into Origami strain of Escherichia coli and expressed after induction with isopropyl thiogalactoside (IPTG). The level of expression was 4 mg/L of culture medium, and the His-tagged recombinant defensin (HisXarJ1-1) was expressed exclusively into inclusion bodies. After solubilization, HisXarJ1-1 was purified by affinity and hydrophobic interaction chromatography. The reverse-phase HPLC profile of the HisXarJ1-1 product obtained from the affinity chromatography step showed single main peptide fraction of molecular masses of 7050.6 Da and after treatment with DTT a single fraction of 7, 042.6 Da corresponding to the reduced peptide was observed. An in vitro folding step of the HisXarJ1-1 generated a distinct profile of oxidized forms of the peptide this oxidized peptide was capable of binding phosphatidic acid in vitro. Possible dimer and oligomer of HisXarJ1-1 were visible in gel electrophoresis and immunodetected with anti-His antibodies. Pure recombinant defensin HisXarJ1-1 exhibited antibacterial activity against Pseudomonas aeruginosa. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Involvement of cathepsin B in mitochondrial apoptosis by p-phenylenediamine under ambient UV radiation.

    Science.gov (United States)

    Goyal, Shruti; Amar, Saroj Kumar; Dubey, Divya; Pal, Manish Kumar; Singh, Jyoti; Verma, Ankit; Kushwaha, Hari Narayan; Ray, Ratan Singh

    2015-12-30

    Paraphenylenediamine (PPD), a derivative of paranitroaniline has been most commonly used as an ingredient of oxidative hair dye and permanent tattoos. We have studied the phototoxic potential of PPD under ambient ultraviolet radiation. PPD is photodegraded and form a novel photoproduct under UV A exposure. PPD shows a concentration dependent decrease in cell viability of human Keratinocyte cells (HaCaT) through MTT and NRU test. Significant intracellular ROS generation was measured by DCFDA assay. It caused an oxidative DNA damage via single stranded DNA breaks, micronuclei and CPD formation. Both lysosome and mitochondria is main target for PPD induced apoptosis which was proved through lysosomal destabilization and release of cathepsin B by immunofluorescence, real time PCR and western blot analysis. Cathepsin B process BID to active tBID which induces the release of cytochrome C from mitochondria. Mitochondrial depolarization was reported through transmission electron microscopy. The cathepsin inhibitor reduced the release of cytochrome C in PPD treated cells. Thus study suggests that PPD leads to apoptosis via the involvement of lysosome and mitochondria both under ambient UV radiation. Therefore, photosensitizing nature of hair dye ingredients should be tested before coming to market as a cosmetic product for the safety of human beings. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Cathepsins: Proteases that are vital for survival but can also be fatal.

    Science.gov (United States)

    Patel, Seema; Homaei, Ahmad; El-Seedi, Hesham R; Akhtar, Nadeem

    2018-06-06

    The state of enzymes in the human body determines the normal physiology or pathology, so all the six classes of enzymes are crucial. Proteases, the hydrolases, can be of several types based on the nucleophilic amino acid or the metal cofactor needed for their activity. Cathepsins are proteases with serine, cysteine, or aspartic acid residues as the nucleophiles, which are vital for digestion, coagulation, immune response, adipogenesis, hormone liberation, peptide synthesis, among a litany of other functions. But inflammatory state radically affects their normal roles. Released from the lysosomes, they degrade extracellular matrix proteins such as collagen and elastin, mediating parasite infection, autoimmune diseases, tumor metastasis, cardiovascular issues, and neural degeneration, among other health hazards. Over the years, the different types and isoforms of cathepsin, their optimal pH and functions have been studied, yet much information is still elusive. By taming and harnessing cathepsins, by inhibitors and judicious lifestyle, a gamut of malignancies can be resolved. This review discusses these aspects, which can be of clinical relevance. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  7. Tumor Necrosis Factor-α Induced Apoptosis in U937 Cells Promotes Cathepsin D-Independent Stefin B Degradation.

    Science.gov (United States)

    Bidovec, Katja; Božič, Janja; Dolenc, Iztok; Turk, Boris; Turk, Vito; Stoka, Veronika

    2017-12-01

    Lysosomal cathepsins were previously found to be involved in tumor necrosis factor-α (TNFα)-induced apoptosis. However, there are opposing views regarding their role as either initiators or amplifiers of the signaling cascade as well as the order of molecular events during this process. In this study, we investigated the role of cathepsin D (catD) in TNFα/cycloheximide-induced apoptosis in U937 human monocytic cells. TNFα-induced apoptosis proceeds through caspase-8 activation, processing of the pro-apoptotic molecule Bid, mitochondrial membrane permeabilization, and caspase-3 activation. The translocation of lysosomal catD into the cytosol was a late event, suggesting that lysosomal membrane permeabilization and the release of cathepsins are not required for the induction of apoptosis, but rather amplifies the process through the generation of reactive oxygen species. For the first time, we show that apoptosis is accompanied by degradation of the cysteine cathepsin inhibitor stefin B (StfB). CatD did not exhibit a crucial role in this step. However, this degradation was partially prevented through pre-incubation with the antioxidant N-acetyl cysteine, although it did not prevent apoptosis and its progression. These results suggest that the degradation of StfB, as a response to TNFα, could induce a cell death amplification effect as a result of progressive damage to lysosomes during TNFα treatment. J. Cell. Biochem. 118: 4813-4820, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  8. Synergistic apoptotic response between valproic acid and fludarabine in chronic lymphocytic leukaemia (CLL) cells involves the lysosomal protease cathepsin B

    International Nuclear Information System (INIS)

    Yoon, J-Y; Szwajcer, D; Ishdorj, G; Benjaminson, P; Xiao, W; Kumar, R; Johnston, J B; Gibson, S B

    2013-01-01

    Fludarabine, a nucleoside analogue, is commonly used in combination with other agents for the treatment of chronic lymphocytic leukaemia (CLL). In previous studies, valproic acid (VPA), an inhibitor of histone deacetylases, combined with fludarabine to synergistically increase apoptotic cell death in CLL cells. In the present study, we found that the combination of fludarabine and VPA decreases the level of the anti-apoptotic proteins Mcl-1 and XIAP in primary CLL cells. Treatment with fludarabine alone, or in combination with VPA, led to the loss of lysosome integrity, and chemical inhibition of the lysosomal protease cathepsin B, using CA074-Me, was sufficient to reduce apoptosis. VPA treatment increased cathepsin B levels and activities in primary CLL cells, thereby priming CLL cells for lysosome-mediated cell death. Six previously treated patients with relapsed CLL were treated with VPA, followed by VPA/fludarabine combination. The combined therapy resulted in reduced lymphocyte count in five out of six and reduced lymph node sizes in four out of six patients. In vivo VPA treatment increased histone-3 acetylation and cathepsin B expression levels. Thus, the synergistic apoptotic response with VPA and fludarabine in CLL is mediated by cathepsin B activation leading to a decrease in the anti-apoptotic proteins

  9. Protein-only, antimicrobial peptide-containing recombinant nanoparticles with inherent built-in antibacterial activity.

    Science.gov (United States)

    Serna, Naroa; Sánchez-García, Laura; Sánchez-Chardi, Alejandro; Unzueta, Ugutz; Roldán, Mónica; Mangues, Ramón; Vázquez, Esther; Villaverde, Antonio

    2017-09-15

    The emergence of bacterial antibiotic resistances is a serious concern in human and animal health. In this context, naturally occurring cationic antimicrobial peptides (AMPs) might play a main role in a next generation of drugs against bacterial infections. Taking an innovative approach to design self-organizing functional proteins, we have generated here protein-only nanoparticles with intrinsic AMP microbicide activity. Using a recombinant version of the GWH1 antimicrobial peptide as building block, these materials show a wide antibacterial activity spectrum in absence of detectable toxicity on mammalian cells. The GWH1-based nanoparticles combine clinically appealing properties of nanoscale materials with full biocompatibility, structural and functional plasticity and biological efficacy exhibited by proteins. Because of the largely implemented biological fabrication of recombinant protein drugs, the protein-based platform presented here represents a novel and scalable strategy in antimicrobial drug design, that by solving some of the limitations of AMPs offers a promising alternative to conventional antibiotics. The low molecular weight antimicrobial peptide GWH1 has been engineered to oligomerize as self-assembling protein-only nanoparticles of around 50nm. In this form, the peptide exhibits potent and broad antibacterial activities against both Gram-positive and Gram-negative bacteria, without any harmful effect over mammalian cells. As a solid proof-of-concept, this finding strongly supports the design and biofabrication of nanoscale antimicrobial materials with in-built functionalities. The protein-based homogeneous composition offer advantages over alternative materials explored as antimicrobial agents, regarding biocompatibility, biodegradability and environmental suitability. Beyond the described prototype, this transversal engineering concept has wide applicability in the design of novel nanomedicines for advanced treatments of bacterial infections

  10. Ligand-receptor assay for evaluation of functional activity of human recombinant VEGF and VEGFR-1 extracellular fragment.

    Science.gov (United States)

    Leopol'd, A V; Baklaushev, V P; Korchagina, A A; Shein, S A; Grinenko, N F; Pavlov, K A; Ryabukhin, I A; Chekhonin, V P

    2012-04-01

    cDNA encoding VEGF and Ig-like extracellular domains 2-4 of VEGFR-1 (sFlt-1(2-4)) were cloned into prokaryotic expression vectors pET32a and pQE60. Recombinant proteins were purified (metal affinity chromatography) and renatured. Chemiluminescent study for the interaction of recombinant VEGF and sFlt-1(2-4) showed that biotinylated VEGF specifically binds to the polystyrene-immobilized receptor extracellular fragment. Biotinylated recombinant sFlt-1 interacts with immobilized VEGF. Analysis of the interaction of immobilized recombinant VEGFR-1 and VEGF with C6 glioma cells labeled with CFDA-SE (vital fluorescent dye) showed that recombinant VEGFR-1 also binds to native membrane-associated VEGF. Recombinant VEGF was shown to bind to specific receptors expressed on the surface of C6 glioma cells. Functional activity of these proteins was confirmed by ligand-receptor assay for VEGF and VEGFR-1 (sFlt-1) and quantitative chemiluminescent detection.

  11. Presence of the propeptide on recombinant lysosomal dipeptidase controls both activation and dimerization.

    Science.gov (United States)

    Dolenc, Iztok; Pain, Roger; Turk, Vito

    2007-01-01

    Lysosomal dipeptidase catalyzes the hydrolysis of dipeptides with unsubstituted terminals. It is a homodimer and binds zinc. Dimerization is an important issue in understanding the enzyme's function. In this study, we investigated the influence of the propeptide on the folding and dimerization of recombinant lysosomal dipeptidase. For this purpose, we separately cloned and overexpressed the mature protein and the proenzyme. The overexpressed proteins were localized exclusively to insoluble inclusion bodies. Refolding of the urea-solubilized inclusion bodies showed that only dipeptidase lacking the propeptide was dimeric. The soluble renatured proenzyme was a monomer, although circular dichroism and fluorescence spectra of the proenzyme indicated the formation of secondary and tertiary structure. The propeptide thus controls dimerization, as well as activation, of lysosomal dipeptidase.

  12. Annealing effects on recombinative activity of nickel at direct silicon bonded interface

    Energy Technology Data Exchange (ETDEWEB)

    Kojima, Takuto, E-mail: tkojima@toyota-ti.ac.jp; Ohshita, Yoshio; Yamaguchi, Masafumi [Toyota Technological Institute, 2-12-1 Hisakata, Tempaku-ku, Nagoya, 468-8511 (Japan)

    2015-09-15

    By performing capacitance transient analyses, the recombination activity at a (110)/(100) direct silicon bonded (DSB) interface contaminated with nickel diffused at different temperatures, as a model of grain boundaries in multicrystalline silicon, was studied. The trap level depth from the valence band, trap density of states, and hole capture cross section peaked at an annealing temperature of 300 °C. At temperatures ⩾400 °C, the hole capture cross section increased with temperature, but the density of states remained unchanged. Further, synchrotron-based X-ray analyses, microprobe X-ray fluorescence (μ-XRF), and X-ray absorption near edge structure (XANES) analyses were performed. The analysis results indicated that the chemical phase after the sample was annealed at 200 °C was a mixture of NiO and NiSi{sub 2}.

  13. Annealing effects on recombinative activity of nickel at direct silicon bonded interface

    International Nuclear Information System (INIS)

    Kojima, Takuto; Ohshita, Yoshio; Yamaguchi, Masafumi

    2015-01-01

    By performing capacitance transient analyses, the recombination activity at a (110)/(100) direct silicon bonded (DSB) interface contaminated with nickel diffused at different temperatures, as a model of grain boundaries in multicrystalline silicon, was studied. The trap level depth from the valence band, trap density of states, and hole capture cross section peaked at an annealing temperature of 300 °C. At temperatures ⩾400 °C, the hole capture cross section increased with temperature, but the density of states remained unchanged. Further, synchrotron-based X-ray analyses, microprobe X-ray fluorescence (μ-XRF), and X-ray absorption near edge structure (XANES) analyses were performed. The analysis results indicated that the chemical phase after the sample was annealed at 200 °C was a mixture of NiO and NiSi 2

  14. Gene trapping in differentiating cell lines: regulation of the lysosomal protease cathepsin B in skeletal myoblast growth and fusion.

    Science.gov (United States)

    Gogos, J A; Thompson, R; Lowry, W; Sloane, B F; Weintraub, H; Horwitz, M

    1996-08-01

    To identify genes regulated during skeletal muscle differentiation, we have infected mouse C2C12 myoblasts with retroviral gene trap vectors, containing a promoterless marker gene with a 5' splice acceptor signal. Integration of the vector adjacent to an actively transcribed gene places the marker under the transcriptional control of the endogenous gene, while the adjacent vector sequences facilitate cloning. The vector insertionally mutates the trapped locus and may also form fusion proteins with the endogenous gene product. We have screened several hundred clones, each containing a trapping vector integrated into a different endogenous gene. In agreement with previous estimates based on hybridization kinetics, we find that a large proportion of all genes expressed in myoblasts are regulated during differentiation. Many of these genes undergo unique temporal patterns of activation or repression during cell growth and myotube formation, and some show specific patterns of subcellular localization. The first gene we have identified with this strategy is the lysosomal cysteine protease cathepsin B. Expression from the trapped allele is upregulated during early myoblast fusion and downregulated in myotubes. A direct role for cathepsin B in myoblast growth and fusion is suggested by the observation that the trapped cells deficient in cathepsin B activity have an unusual morphology and reduced survival in low-serum media and undergo differentiation with impaired cellular fusion. The phenotype is reproduced by antisense cathepsin B expression in parental C2C12 myoblasts. The cellular phenotype is similar to that observed in cultured myoblasts from patients with I cell disease, in which there is diminished accumulation of lysosomal enzymes. This suggests that a specific deficiency of cathepsin B could contribute to the myopathic component of this illness.

  15. Formation of tissue factor activity following incubation of recombinant human tissue factor apoprotein with plasma lipoproteins

    International Nuclear Information System (INIS)

    Sakai, T.; Kisiel, W.

    1990-01-01

    Incubation of recombinant human tissue factor apoprotein (Apo-TF) with human plasma decreased the recalcified clotting time of this plasma in a time-and dose-dependent manner suggesting relipidation of the Apo-TF by plasma lipoproteins. Incubation of Apo-TF with purified preparations of human very low density, low density and high density lipoproteins resulted in tissue factor activity in a clotting assay. The order of effectiveness was VLDL greater than LDL much greater than HDL. Tissue factor activity generated by incubation of a fixed amount of Apo-TF with plasma lipoproteins was lipoprotein concentration-dependent and saturable. The association of Apo-TF with lipoprotein particles was supported by gel filtration studies in which 125 I-Apo-TF coeluted with the plasma lipoprotein in the void volume of a Superose 6 column in the presence and absence of calcium ions. In addition, void-volume Apo-TF-lipoprotein fractions exhibited tissue factor activity. These results suggest that the factor VIII-bypassing activity of bovine Apo-TF observed in a canine hemophilic model may be due, in part, to its association with plasma lipoproteins and expression of functional tissue factor activity

  16. Molecular Imaging of Cancer Using X-ray Computed Tomography with Protease Targeted Iodinated Activity-Based Probes.

    Science.gov (United States)

    Gaikwad, Hanmant K; Tsvirkun, Darya; Ben-Nun, Yael; Merquiol, Emmanuelle; Popovtzer, Rachela; Blum, Galia

    2018-03-14

    X-ray computed tomography (CT) is a robust, precise, fast, and reliable imaging method that enables excellent spatial resolution and quantification of contrast agents throughout the body. However, CT is largely inadequate for molecular imaging applications due mainly to its low contrast sensitivity that forces the use of large concentrations of contrast agents for detection. To overcome this limitation, we generated a new class of iodinated nanoscale activity-based probes (IN-ABPs) that sufficiently accumulates at the target site by covalently binding cysteine cathepsins that are exceptionally highly expressed in cancer. The IN-ABPs are comprised of a short targeting peptide selective to specific cathepsins, an electrophilic moiety that allows activity-dependent covalent binding, and tags containing dendrimers with up to 48 iodine atoms. IN-ABPs selectively bind and inhibit activity of recombinant and intracellular cathepsin B, L, and S. We compared the in vivo kinetics, biodistribution, and tumor accumulation of IN-ABPs bearing 18 and 48 iodine atoms each, and their control counterparts lacking the targeting moiety. Here we show that although both IN-ABPs bind specifically to cathepsins within the tumor and produce detectable CT contrast, the 48-iodine bearing IN-ABP was found to be optimal with signals over 2.1-fold higher than its nontargeted counterpart. In conclusion, this study shows the synthetic feasibility and potential utility of IN-ABPs as potent contrast agents that enable molecular imaging of tumors using CT.

  17. Construction of a plasmid coding for green fluorescent protein tagged cathepsin L and data on expression in colorectal carcinoma cells

    Directory of Open Access Journals (Sweden)

    Tripti Tamhane

    2015-12-01

    Full Text Available The endo-lysosomal cysteine cathepsin L has recently been shown to have moonlighting activities in that its unexpected nuclear localization in colorectal carcinoma cells is involved in cell cycle progression (Tamhane et al., 2015 [1]. Here, we show data on the construction and sequence of a plasmid coding for human cathepsin L tagged with an enhanced green fluorescent protein (phCL-EGFP in which the fluorescent protein is covalently attached to the C-terminus of the protease. The plasmid was used for transfection of HCT116 colorectal carcinoma cells, while data from non-transfected and pEGFP-N1-transfected cells is also shown. Immunoblotting data of lysates from non-transfected controls and HCT116 cells transfected with pEGFP-N1 and phCL-EGFP, showed stable expression of cathepsin L-enhanced green fluorescent protein chimeras, while endogenous cathepsin L protein amounts exceed those of hCL-EGFP chimeras. An effect of phCL-EGFP expression on proliferation and metabolic states of HCT116 cells at 24 h post-transfection was observed.

  18. Long-term stability of recombinant tissue plasminogen activator at -80 C

    Directory of Open Access Journals (Sweden)

    Sperling Matthew

    2009-06-01

    Full Text Available Abstract Background Recombinant tissue plasminogen activator (tPA is a thrombolytic widely used clinically in the treatment of acute thrombotic disease such as ischemic stroke, myocardial infarction, and deep venous thrombosis. This has led to much interest in tPA based lytic therapies leading to laboratory based in-vitro and in-vivo investigations using this drug. However, tPA reconstituted in solution exhibits full activity for only 6–8 hours, according to the manufacturer. Therefore, methods to store reconstituted tPA for long durations while maintaining activity would be of assistance to laboratories using this enzyme. Findings In this work, the enzymatic activity of tPA stored at -80 C over time was measured, using an ELISA technique that measured the amount of active tPA bound to plasminogen activator inhibitor 1 (PAI-1 in a given sample. Sample of tPA solution mixed to a concentration of 1 (mg/ml were stored in cryogenic vials at -80 C for up to 7 years. For a given sample, aliquots were assayed for tPA activity, and compared with a tPA standard to determine relative enzymatic activity. Results are reported as means with standard errors, and 12 measurements were performed for each sample age. Conclusion There was no decrease in tPA activity for samples stored up to 7 years. Such cryogenic storage is a viable method for the preservation of tPA solution for laboratory investigations of tPA-based lytic therapies.

  19. Upregulation of cathepsin S in psoriatic keratinocytes.

    Science.gov (United States)

    Schönefuss, Alexander; Wendt, Wiebke; Schattling, Benjamin; Schulten, Roxane; Hoffmann, Klaus; Stuecker, Markus; Tigges, Christian; Lübbert, Hermann; Stichel, Christine

    2010-08-01

    Cathepsin S (CATS) is a cysteine protease, well known for its role in MHC class II-mediated antigen presentation and extracellular matrix degradation. Disturbance of the expression or metabolism of this protease is a concomitant feature of several diseases. Given this importance we studied the localization and regulation of CATS expression in normal and pathological human/mouse skin. In normal human skin CATS-immunostaining is mainly present in the dermis and is localized in macrophages, Langerhans, T- and endothelial cells, but absent in keratinocytes. In all analyzed pathological skin biopsies, i.e. atopic dermatitis, actinic keratosis and psoriasis, CATS staining is strongly increased in the dermis. But only in psoriasis, CATS-immunostaining is also detectable in keratinocytes. We show that cocultivation with T-cells as well as treatment with cytokines can trigger expression and secretion of CATS, which is involved in MHC II processing in keratinocytes. Our data provide first evidence that CATS expression (i) is selectively induced in psoriatic keratinocytes, (ii) is triggered by T-cells and (iii) might be involved in keratinocytic MHC class II expression, the processing of the MHC class II-associated invariant chain and remodeling of the extracellular matrix. This paper expands our knowledge on the important role of keratinocytes in dermatological disease.

  20. Dendritic cell activation and maturation induced by recombinant calreticulin fragment 39-272.

    Science.gov (United States)

    Li, Yue; Zeng, Xiaoli; He, Lijuan; Yuan, Hui

    2015-01-01

    Dendritic cells (DC) are the most potent antigen-presenting cells for initiating immune responses. DC maturation can be induced by exposing of immature DC to pathogen products or pro-inflammatory factor, which dramatically enhances the ability of DC to activate Ag-specific T cells. In this study, a recombinant calreticulin fragment 39-272 (rCRT/39-272) covering the lectin-like N domain and partial P domain of murine CRT has been expressed and purified in Escherichia coli. Functional analysis studies revealed that rCRT/39-272 has potent immunostimulatory activities in both activating human monocytes and B cells to secrete cytokines. rCRT/39-272 can drive the activation of bone marrow derived DC in TLR4/CD14 dependent way, as indicated by secretion of cytokines IL-12/IL-23 (p40) and IL-1β. Exposure of DC to rCRT/39-272 induces P-Akt, suggesting that rCRT/39-272 induces maturation of DC through PI3K/Akt signaling pathway. The results suggest that soluble rCRT/39-272 is a potent stimulatory agent to DC maturation in TLR4/CD14 and PI3K/Akt dependent pathway. It may play important roles in initiating cellular immunity in vivo and the T cell response in vitro. Thus it could be used for study of DC-based tumor vaccines.

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

    NARCIS (Netherlands)

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

    2015-01-01

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

  2. Functional properties of the recombinant kringle-2 domain of tissue plasminogen activator produced in Escherichia coli

    International Nuclear Information System (INIS)

    Wilhelm, O.G.; Jaskunas, S.R.; Vlahos, C.J.; Bang, N.U.

    1990-01-01

    The kringle-2 domain (residues 176-262) of tissue-type plasminogen activator (t-PA) was cloned and expressed in Escherichia coli. The recombinant peptide, which concentrated in cytoplasmic inclusion bodies, was isolated, solubilized, chemically refolded, and purified by affinity chromatography on lysine-Sepharose to apparent homogeneity. [35S]Cysteine-methionine-labeled polypeptide was used to study the interactions of kringle-2 with lysine, fibrin, and plasminogen activator inhibitor-1. The kringle-2 domain bound to lysine-Sepharose and to preformed fibrin with a Kd = 104 +/- 6.2 microM (0.86 +/- 0.012 binding site) and a Kd = 4.2 +/- 1.05 microM (0.80 +/- 0.081 binding site), respectively. Competition experiments and direct binding studies showed that the kringle-2 domain is required for the formation of the ternary t-PA-plasminogen-intact fibrin complex and that the association between the t-PA kringle-2 domain and fibrin does not require plasmin degradation of fibrin and exposure of new COOH-terminal lysine residues. We also observed that kringle-2 forms a complex with highly purified guanidine-activated plasminogen activator inhibitor-1, dissociable by 0.2 M epsilon-aminocaproic acid. The kringle-2 polypeptide significantly inhibited tissue plasminogen activator/plasminogen activator inhibitor-1 interaction. The kringle-2 domain bound to plasminogen activator inhibitor-1 in a specific and saturable manner with a Kd = 0.51 +/- 0.055 microM (0.35 +/- 0.026 binding site). Therefore, the t-PA kringle-2 domain is important for the interaction of t-PA not only with fibrin, but also with plasminogen activator inhibitor-1 and thus represents a key structure in the regulation of fibrinolysis

  3. Residue-specific annotation of disorder-to-order transition and cathepsin inhibition of a propeptide-like crammer from D. melanogaster.

    Directory of Open Access Journals (Sweden)

    Tien-Sheng Tseng

    Full Text Available Drosophila melanogaster crammer is a novel cathepsin inhibitor involved in long-term memory formation. A molten globule-to-ordered structure transition is required for cathepsin inhibition. This study reports the use of alanine scanning to probe the critical residues in the two hydrophobic cores and the salt bridges of crammer in the context of disorder-to-order transition and cathepsin inhibition. Alanine substitution of the aromatic residues W9, Y12, F16, Y20, Y32, and W53 within the hydrophobic cores, and charged residues E8, R28, R29, and E67 in the salt bridges considerably decrease the ability of crammer to inhibit Drosophila cathepsin B (CTSB. Far-UV circular dichroism (CD, intrinsic fluorescence, and nuclear magnetic resonance (NMR spectroscopies show that removing most of the aromatic and charged side-chains substantially reduces thermostability, alters pH-dependent helix formation, and disrupts the molten globule-to-ordered structure transition. Molecular modeling indicates that W53 in the hydrophobic Core 2 is essential for the interaction between crammer and the prosegment binding loop (PBL of CTSB; the salt bridge between R28 and E67 is critical for the appropriate alignment of the α-helix 4 toward the CTSB active cleft. The results of this study show detailed residue-specific dissection of folding transition and functional contributions of the hydrophobic cores and salt bridges in crammer, which have hitherto not been characterized for cathepsin inhibition by propeptide-like cysteine protease inhibitors. Because of the involvements of cathepsin inhibitors in neurodegenerative diseases, these structural insights can serve as a template for further development of therapeutic inhibitors against human cathepsins.

  4. Identification of interleukin-8 converting enzyme as cathepsin L.

    Science.gov (United States)

    Ohashi, Kensaku; Naruto, Masanobu; Nakaki, Toshio; Sano, Emiko

    2003-06-26

    IL-8 is produced by various cells, and the NH(2)-terminal amino acid sequence of IL-8 displays heterogeneity among cell types. The mature form of IL-8 has 72 amino acids (72IL-8), while a precursor form (77IL-8) of IL-8 has five additional amino acids to the 72IL-8 NH(2)-terminal. However, it has been unclear how IL-8 is processed to yield the mature form. In this study, converting enzyme was purified as a single 31-kDa band on silver-stained polyacrylamide gel from 160 l of cultured fibroblast supernatant by sequential chromatography. NH(2)-terminal amino acid sequence analysis revealed a sequence, EAPRSVDWRE, which was identified as a partial sequence of cathepsin L. Polyclonal antibodies raised against cathepsin L recognized the purified converting enzyme on Western blot. Moreover, human hepatic cathepsin L cleaved 77IL-8 between Arg(5) and Ser(6), which is the same cleavage site as the putative converting enzyme, resulting in 72IL-8 formation. These data indicate that the converting enzyme of the partially purified fraction of the human fibroblast culture supernatant was cathepsin L. Furthermore, 72IL-8 was sevenfold more potent than 77IL-8 in a neutrophil chemotaxis assay. These results show that cathepsin L is secreted from human fibroblasts in response to external stimuli and plays an important role in IL-8 processing in inflammatory sites.

  5. Recombinant activated factor VII: its mechanism of action and role in the control of hemorrhage.

    Science.gov (United States)

    Allen, Geoffrey A; Hoffman, Maureane; Roberts, Harold R; Monroe, Dougald M

    2002-12-01

    Recombinant activated factor VII (rFVIIa) has proven both safe and efficacious in the treatment of bleeding episodes in patients with hemophilia A or B who have developed inhibitors. More recently, a growing number of reports suggests that rFVIIa may also have indications for the treatment of bleeding in patients with other hemostatic disorders, including qualitative and quantitative platelet defects, factor deficiencies other than hemophilia, and in otherwise healthy patients with uncontrollable hemorrhage following surgery or trauma. We have attempted to reconcile the various proposed mechanisms of action of rFVIIa with its apparent efficacy in such diverse clinical settings. A review of the literature was performed to determine those clinical scenarios in which rFVIIa appears to have been effective in controlling associated hemorrhage. Findings from our group and others have demonstrated that rFVIIa is able to directly activate factor X and increase thrombin production on the surface of activated platelets in the absence of factor VIII or IX, as well as to improve thrombin generation in thrombocytopenia, and to yield a fibrin dot more resistant to fibrinolysis in vitro. Through these primary mechanisms, we believe that rFVIIa may be able to compensate for a variety of defects in hemostasis and merits further investigation as a general therapeutic for uncontrollable hemorrhage.

  6. Expression and Localization of Cathepsins B, D, and G in Dupuytren’s Disease

    Directory of Open Access Journals (Sweden)

    Kirin Tan, MB ChB

    2018-02-01

    Conclusions:. Cathepsins B, D, and G were expressed in the DD tissues, with cathepsins B and D localized to the primitive population in the endothelium of the microvessels, whereas cathepsin G was localized to phenotypic mast cells, suggesting the presence of bypass loops for the RAS.

  7. Localization profile of Cathepsin L in the brain of African giant rat ...

    African Journals Online (AJOL)

    Cathepsins, are members of the papain superfamily of mammalian lysosomal cysteine proteases. Among others there are two prominent members with broad substrate specificity, these are cathepsin B and cathepsin L that are known to be involved in the process of intra- and extra-cellular protein degradation and turnover.

  8. Treatment of massive gastrointestinal bleeding occurred during autologous stem cell transplantation with recombinant activated factor VII and octreotide

    Directory of Open Access Journals (Sweden)

    Erman Atas

    2015-01-01

    Full Text Available After hematopoietic stem cell transplantation (HSCT, patients may suffer from bleeding. One of the bleeding type is gastrointestinal (GI which has serious morbidity and mortality in children with limited treatment options. Herein, we presented a child with upper GI bleeding post autologous HSCT controlled successfully by using recombinant activated factor VII (rFVIIa and octreotide infusion.

  9. Increased biological activity of deglycosylated recombinant human granulocyte/macrophage colony-stimulating factor produced by yeast or animal cells

    International Nuclear Information System (INIS)

    Moonen, P.; Mermod, J.J.; Ernst, J.F.; Hirschi, M.; DeLamarter, J.F.

    1987-01-01

    Human granulocyte/macrophage colony-stimulating factor (hGM-CSF) produced by several recombinant sources including Escherichia coli, yeast, and animal cells was studied. Recombinant animal cells produced hGM-CSF in low quantities and in multiple forms of varying size. Mammalian hGM-CSF was purified 200,000-fold using immunoaffinity and lectin chromatography. Partially purified proteins produced in yeast and mammalian cells were assayed for the effects of deglycosylation. Following enzymatic deglycosylation, immunoreactivity was measured by radioimmunoassay and biological activity was measured in vitro on responsive human primary cells. Removal of N-linked oligosaccharides from both proteins increased their immunoreactivities by 4- to 8-fold. Removal of these oligosaccharides also increased their specific biological activities about 20-fold, to reach approximately the specific activity of recombinant hGM-CSF from E. coli. The E. coli produced-protein-lacking any carbohydrate- had by far the highest specific activity observed for the recombinant hGM-CSFs

  10. Activation of human T cells by a tumor vaccine infected with recombinant Newcastle disease virus producing IL-2

    NARCIS (Netherlands)

    Janke, M.; Peeters, B.; Zhao, H.; Leeuw, O.; Moormann, R.J.M.; Arnold, A.; Ziouta, Y.; Fournier, P.; Schirrmacher, V.

    2008-01-01

    A new recombinant (rec) Newcastle disease virus (NDV) with incorporated human interleukin 2 (IL-2) as foreign therapeutic gene [rec(IL-2)] will be described. The foreign gene in rec(IL-2) did not affect the main features of NDV replication nor its tumor selectivity. Biologically active IL-2 was

  11. Recombinant tissue plasminogen activator as a novel treatment option for infective endocarditis: a retrospective clinical study in 32 children.

    Science.gov (United States)

    Levitas, Aviva; Krymko, Hanna; Richardson, Justin; Zalzstein, Eli; Ioffe, Viktoriya

    2016-01-01

    Infective endocarditis is a life-threatening infectious syndrome, with high morbidity and mortality. Current treatments for infective endocarditis include intravenous antibiotics, surgery, and involve a lengthy hospital stay. We hypothesised that adjunctive recombinant tissue plasminogen activator treatment for infective endocarditis may facilitate faster resolution of vegetations and clearance of positive blood cultures, and therefore decrease morbidity and mortality. This retrospective study included follow-up of patients, from 1997 through 2014, including clinical presentation, causative organism, length of treatment, morbidity, and mortality. We identified 32 patients, all of whom were diagnosed with endocarditis and were treated by recombinant tissue plasminogen activator. Among all, 27 patients (93%) had positive blood cultures, with the most frequent organisms being Staphylococcus epidermis (nine patients), Staphylococcus aureus (six patients), and Candida (nine patients). Upon treatment, in 31 patients (97%), resolution of vegetations and clearance of blood cultures occurred within hours to few days. Out of 32 patients, one patient (3%) died and three patients (9%) suffered embolic or haemorrhagic events, possibly related to the recombinant tissue plasminogen activator. None of the patients required surgical intervention to assist vegetation resolution. In conclusion, it appears that recombinant tissue plasminogen activator may become an adjunctive treatment for infective endocarditis and may decrease morbidity as compared with current guidelines. Prospective multi-centre studies are required to validate our findings.

  12. Bioconjugation of recombinant tissue plasminogen activator to magnetic nanocarriers for targeted thrombolysis

    Directory of Open Access Journals (Sweden)

    Yang HW

    2012-10-01

    Full Text Available Hung-Wei Yang,1,* Mu-Yi Hua,1,* Kun-Ju Lin,2,* Shiaw-Pyng Wey,3 Rung-Ywan Tsai,4 Siao-Yun Wu,5 Yi-Ching Lu,5 Hao-Li Liu,6 Tony Wu,7 Yunn-Hwa Ma5 1Chang Gung Molecular Medicine Research Center, Department of Chemical and Materials Engineering, 2Molecular Imaging Center, Department of Nuclear Medicine, Chang Gung Memorial Hospital, Kuei-Shan, Tao-Yuan, Taiwan, Republic of China; 3Department of Medical Imaging and Radiological Sciences, 4Electronics and Optoelectronics Research Laboratories, Industrial Technology Research Institute, Hsin-chu, Taiwan, Republic of China; 5Department of Physiology and Pharmacology and Healthy Aging Research Center, 6Department of Electrical Engineering, Chang Gung University, Kuei-Shan, Tao-Yuan, Taiwan, Republic of China; 7Department of Neurology, Chang Gung University College of Medicine and Memorial Hospital, Tao-Yuan, Taiwan, Republic of China*These authors contributed equally to this workAbstract: Low-toxicity magnetic nanocarriers (MNCs composed of a shell of poly [aniline-co-N-(1-one-butyric acid aniline] over a Fe3O4 magnetic nanoparticle core were developed to carry recombinant tissue plasminogen activator (rtPA in MNC-rtPA for targeted thrombolysis. With an average diameter of 14.8 nm, the MNCs exerted superparamagnetic properties. Up to 276 µg of active rtPA was immobilized per mg of MNCs, and the stability of the immobilized rtPA was greatly improved during storage at 4°C and 25°C. In vitro thrombolysis testing with a tubing system demonstrated that magnet-guided MNC-rtPA showed significantly improved thrombolysis compared with free rtPA and reduced the clot lysis time from 39.2 ± 3.2 minutes to 10.8 ± 4.2 minutes. In addition, magnet-guided MNC-rtPA at 20% of the regular rtPA dose restored blood flow within 15–25 minutes of treatment in a rat embolism model without triggering hematological toxicity. In conclusion, this improved system is based on magnetic targeting accelerated thrombolysis and is

  13. Influence of natural and recombinant interferons on development of antiviral condition and activity of natural killers

    International Nuclear Information System (INIS)

    Kuznetsov, V.P.; Avdeev, G.I.; Vyadro, M.M.; Leikin, Yu.D.; Frolova, I.S.

    1986-01-01

    For the purpose of a preliminary estimate of the therapeutic potential of domestic recombinant alpha 2 -component of human leukocytic interferon (rl) in vitro tests, the authors studied its ability to induce development of antiviral condition in diploid culture of human embryo fibroblasts and to activate the cytolytic effect of natural killers in relation to tumor cells, of the K-562 leukemia line and cells of lung adenocarcinoma. The authors used a medicinal form of rL which was derived by expression of a reconstructed gene in Escherichia coli cells. Part of the tests were conducted with an analogous preparation synthesized using another producer, Pseudomonas sp). The biological effect of both preparations was the same. For comparison, a natural preparation was used in all tests: human leukocytic interferon for injection, II(le). The authors studied activity of natural killers in a fraction of mononuclears isolated from blood of essentially healthy donors and from cancer patients. Cells were incubated for 2 h with various concentrations of interferons, then combined in a ratio of 25-50:1 with target cells labeled with 51 Cr. Cytotoxic reaction was conducted for 4 (4-CTR) or 18 h (18-CTR) at 37 0 C. Natural killers could thus be divided into two subpopulations: killer (4-CTR) and cytotoxic (18-CTR) cells. In preliminary tests, both preparations possessed the ability to active natural killers. The effective concentration for rL was within the limits of 1000-2000 IU/ml, and 50-200 Iu/ml for Le. The data on activation of natural killers in 16 oncological patients (primarily with lung cancer), the authors established that both rL and Le induced activation of natural killers in the overwhelming majority of cases in relation to K-562 target cells and adenocarcinomas of the lung

  14. Restricted expression of recombination activating gene (RAG-1) in mouse lymphoid tissues

    International Nuclear Information System (INIS)

    Yamamoto, Akihito; Fujinaga, Hiroyuki; Hamatani, Kiyohiro; Atsuta, Mitsuru.

    1993-03-01

    In an attempt to determine the distribution of recombinase activity in the mouse thymus, spleen, and lymph nodes, we used the in situ hybridization method to examine the expression of the recombination activating genes RAG-1 and RAG-2. Expression of RAG-1 was found in most cortical thymocytes but not in the majority of medullary thymocytes. Although hybridization signals of RAG-2 were not as intense as those of RAG-1, the localization of RAG-2 transcripts was similar to that of RAG-1. In the spleen, expression of RAG-1 was found only in limited cells near the splenic sinus, and the majority of the cells within the follicle were negative for RAG-1 transcript. In nude mice, RAG-1-expressing cells were detected in the same regions, which suggests that in situ hybridization signals of RAG-1 in the spleen are due to the cells of B-cell origin. In the lymph nodes, expression of RAG-1 was found only in the medullary region. Expression of RAG-2 transcript in the spleen and the lymph nodes, if any, was too faint to allow determination of the specific localization. These results suggest that most of the cortical thymocytes and some cells in the spleen are capable of rearranging T-cell receptor genes and immunoglobulin genes, respectively, but the possible involvement of the RAG-1 transcript in RAG-1-positive cells of the spleen and the lymph nodes in functions other than the rearrangement of genes could not be ruled out. (author)

  15. Expression, Purification and Bioactivities Analysis of Recombinant Active Peptide from Shark Liver

    Directory of Open Access Journals (Sweden)

    Boping Ye

    2009-06-01

    Full Text Available The Active Peptide from Shark Liver (APSL was expressed in E. coli BL21 cells. The cDNA encoding APSL protein was obtained from shark regenerated hepatic tissue by RT-PCR, then it was cloned in the pET-28a expression vector. The expressed fusion protein was purified by Ni-IDA affinity chromatography. SDS-PAGE and HPLC analysis showed the purity of the purified fusion protein was more than 98%. The recombinant APSL (rAPSL was tested for its biological activity both in vitro, by its ability to improve the proliferation of SMMC7721 cells, and in vivo, by its significant protective effects against acute hepatic injury induced by CCl4 and AAP (acetaminophen in mice. In addition, the rAPSL could decrease the blood glucose concentration of mice with diabetes mellitus induced by alloxan. Paraffin sections of mouse pancreas tissues showed that rAPSL (3 mg/kg could effectively protect mouse islets from lesions induced by alloxan, which indicated its potential application in theoretical research and industry.

  16. PRDM9 variation strongly influences recombination hot-spot activity and meiotic instability in humans.

    Science.gov (United States)

    Berg, Ingrid L; Neumann, Rita; Lam, Kwan-Wood G; Sarbajna, Shriparna; Odenthal-Hesse, Linda; May, Celia A; Jeffreys, Alec J

    2010-10-01

    PRDM9 has recently been identified as a likely trans regulator of meiotic recombination hot spots in humans and mice. PRDM9 contains a zinc finger array that, in humans, can recognize a short sequence motif associated with hot spots, with binding to this motif possibly triggering hot-spot activity via chromatin remodeling. We now report that human genetic variation at the PRDM9 locus has a strong effect on sperm hot-spot activity, even at hot spots lacking the sequence motif. Subtle changes within the zinc finger array can create hot-spot nonactivating or enhancing variants and can even trigger the appearance of a new hot spot, suggesting that PRDM9 is a major global regulator of hot spots in humans. Variation at the PRDM9 locus also influences aspects of genome instability-specifically, a megabase-scale rearrangement underlying two genomic disorders as well as minisatellite instability-implicating PRDM9 as a risk factor for some pathological genome rearrangements.

  17. Cre Activated and Inactivated Recombinant Adeno-Associated Viral Vectors for Neuronal Anatomical Tracing or Activity Manipulation.

    Science.gov (United States)

    Saunders, Arpiar; Sabatini, Bernardo L

    2015-07-01

    Recombinant adeno-associated viruses (rAAVs) transcriptionally activated by Cre recombinase (Cre-On) are powerful tools for determining the anatomy and function of genetically defined neuronal types in transgenic Cre driver mice. Here we describe how rAAVs transcriptionally inactivated by Cre (Cre-Off) can be used in conjunction with Cre-On rAAVs or genomic Cre-reporter alleles to study brain circuits. Intracranial injection of Cre-On/Cre-Off rAAVs into spatially intermingled Cre(+) and Cre(-) neurons allows these populations to be differentially labeled or manipulated within individual animals. This comparison helps define the unique properties of Cre(+) neurons, highlighting the specialized role they play in their constituent brain circuits. This protocol touches on the conceptual and experimental background of Cre-Off rAAV systems, including caveats and methods of validation. Copyright © 2015 John Wiley & Sons, Inc.

  18. Cathepsin-D And Tnf-α in Bladder Cancer

    Directory of Open Access Journals (Sweden)

    T. Salman

    1996-01-01

    Full Text Available In a study of 34 normal healthy controls, 35 patients with urinary tract bilharziasis and 93 bladder cancer patients (62 of them are operable cases and 31 are non-operable ones, serum tumor necrosis factor alpha (TNF-α and cytosolic Cathepsin-D were estimated. Though both potential markers were elevated in bladder cancer patients, neither Cathepsin-D nor TNF-α showed associations of prognostic value since there were no positive correlations with tumor stages, grades or association of tumors with bilharzia ova or lymph node involvement.

  19. In vitro effects of recombinant activated factor VII on thrombin generation and coagulation following inhibition of platelet procoagulant activity by prasugrel.

    Science.gov (United States)

    Mazzeffi, Michael; Szlam, Fania; Jakubowski, Joseph A; Tanaka, Kenichi A; Sugidachi, Atsuhiro; Levy, Jerrold H

    2013-07-01

    Prasugrel is a thienopyridyl P2Y12 antagonist with potent antiplatelet effects. At present, little is known about its effects on thrombin generation or what strategies may emergently reverse its anticoagulant effects. In the current study we evaluated whether recombinant activated factor VII may reverse prasugrel induced effects and increase thrombin generation in an in vitro model. The effect of prasugrel active metabolite, PAM (R-138727), was evaluated on platelet aggregation, thrombin generation, and rotational thromboelastometry parameters using blood from 20 healthy volunteers. Additionally, we evaluated the effects of adenosine diphosphate (ADP) and recombinant activated factor VII on restoring these parameters towards baseline values. PAM reduced maximum platelet aggregation and led to platelet disaggregation. It also decreased peak thrombin, increased lag time, and increased time to peak thrombin. Treatment with recombinant activated factor VII restored all three parameters of thrombin generation towards baseline. ADP decreased lag time and time to peak thrombin, but had no effect on peak thrombin. When recombinant activated factor VII and ADP were combined they had a greater effect on thrombin parameters than either drug alone. PAM also increased thromboelastometric clotting time and clot formation time, but had no effect on maximum clot firmness. Treatment with either recombinant activated factor VII or ADP restored these values towards baseline. Recombinant activated factor VII restores thrombin generation in the presence of PAM. In patients taking prasugrel with life-threatening refractory bleeding it has the potential to be a useful therapeutic approach. Additional clinical studies are needed to validate our findings. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Analysis of genotoxic activity of ketamine and rocuronium bromide using the somatic mutation and recombination test in Drosophila melanogaster.

    Science.gov (United States)

    Koksal, Pakize Muge; Gürbüzel, Mehmet

    2015-03-01

    The present study evaluated the mutagenic and recombinogenic effects of two commonly used anesthetic agents, ketamine and rocuronium bromide, in medicine using the wing somatic mutation and recombination test (SMART) in Drosophila. The standard (ST) cross and the high-bioactivation (HB) cross with high sensitivity to procarcinogens and promutagens were used. The SMART test is based on the loss of heterozygosity, which occurs via various mechanisms, such as chromosome loss and deletion, half-translocation, mitotic recombination, mutation, and non-disjunction. Genetic alterations occurring in the somatic cells of the wing's imaginal discs result in mutant clones in the wing blade. Three-day-old trans-heterozygous larvae with two recessive markers, multiple wing hairs (mwh) and flare (flr(3)), were treated with ketamine and rocuronium bromide. Analysis of the ST cross indicated that ketamine exhibited genotoxicity activity and that this activity was particularly dependent on homologous mitotic recombination at concentrations of 250 μg/ml and above. Rocuronium bromide did not exert mutagenic and/or recombinogenic effects. In the HB cross, ketamine at a concentration of 1000 μg/ml and rocuronium bromide at all concentrations, with the exception of 250 μg/ml (inconclusive), exerted genotoxic effects, which could also be associated with the increase in mitotic recombination. Copyright © 2015 Elsevier B.V. All rights reserved.

  1. Evidence supporting the use of recombinant activated factor VII in congenital bleeding disorders

    Directory of Open Access Journals (Sweden)

    Pär I Johansson

    2010-06-01

    Full Text Available Pär I Johansson, Sisse R OstrowskiCapital Region Blood Bank, Section for Transfusion Medicine, Rigshospitalet, University of Copenhagen, Copenhagen, DenmarkBackground: Recombinant activated factor VII (rFVIIa, NovoSeven® was introduced in 1996 for the treatment of hemophilic patients with antibodies against coagulation factor VIII or IX.Objective: To review the evidence supporting the use of rFVIIa for the treatment of patients with congenital bleeding disorders.Patients and methods: English-language databases were searched in September 2009 for reports of randomized controlled trials (RCTs evaluating the ability of rFVIIa to restore hemostasis in patients with congenital bleeding disorders.Results: Eight RCTs involving 256 hemophilic patients with antibodies against coagulation factors, also known as inhibitors, were identified. The evidence supporting the use of rFVIIa in these patients was weak with regard to dose, clinical setting, mode of administration, efficacy, and adverse events, given the limited sample size of each RCT and the heterogeneity of the studies.Conclusion: The authors suggest that rFVIIa therapy in hemophilic patients with inhibitors should be based on the individual’s ability to generate thrombin and form a clot, and not on the patient’s weight alone. Therefore, assays for thrombin generation, such as whole-blood thromboelastography, have the potential to significantly improve the treatment of these patients.Keywords: hemophilia, inhibitors, coagulation factor VIII, coagulation factor IX, rFVIIa, NovoSeven, FEIBA, hemostasis, RCT

  2. Novel recombinant adeno-associated viruses for Cre activated and inactivated transgene expression in neurons

    Science.gov (United States)

    Saunders, Arpiar; Johnson, Caroline A.; Sabatini, Bernardo L.

    2012-01-01

    Understanding the organization of the nervous system requires methods for dissecting the contributions of each component cell type to circuit function. One widely used approach combines genetic targeting of Cre recombinase to specific cell populations with infection of recombinant adeno-associated viruses (rAAVs) whose transgene expression is activated by Cre (“Cre-On”). Distinguishing how the Cre-expressing neurons differ functionally from neighboring Cre-negative neurons requires rAAVs that are inactivated by Cre (“Cre-Off”) and can be used in tandem with Cre-On viruses. Here we introduce two rAAV vectors that are inactivated by Cre and carry different fluorophore and optogenetic constructs. We demonstrate single and dual rAAV systems to achieve Cre-On and Cre-Off expression in spatially-intermingled cell populations of the striatum. Using these systems, we uncovered cryptic genomic interactions that occur between multiple Cre-sensitive rAAVs or between Cre-sensitive rAAVs and somatic Cre-conditional alleles and devised methods to avoid these interactions. Our data highlight both important experimental caveats associated with Cre-dependent rAAV use as well as opportunities for the development of improved rAAVs for gene delivery. PMID:22866029

  3. Constitutive cellulase production from glucose using the recombinant Trichoderma reesei strain overexpressing an artificial transcription activator.

    Science.gov (United States)

    Zhang, Xiaoyue; Li, Yonghao; Zhao, Xinqing; Bai, Fengwu

    2017-01-01

    The high cost of cellulase production presents biggest challenge in biomass deconstruction. Cellulase production by Trichoderma reesei using low cost carbon source is of great interest. In this study, an artificial transcription activator containing the Cre1 binding domain linked to the Xyr1 effector and binding domains was designed and constitutively overexpressed in T. reesei RUT C30. The recombinant strain T. reesei zxy-2 displayed constitutive cellulase production using glucose as a sole carbon source, and the production titer was 12.75-fold of that observed with T. reesei RUT C30 in shake flask culture. Moreover, FPase and xylanase titers of 2.63 and 108.72IU/mL, respectively, were achieved using glucose as sole carbon source within 48h in a 7-L fermenter by batch fermentation using T. reesei zxy-2. The crude enzyme obtained was used to hydrolyze alkali pretreated corn stover, and a high glucose yield of 99.18% was achieved. Copyright © 2016. Published by Elsevier Ltd.

  4. Recombinant interleukin 2 stimulates in vivo proliferation of adoptively transferred lymphokine-activated killer (LAK) cells

    International Nuclear Information System (INIS)

    Ettinghausen, S.E.; Lipford, E.H. III; Mule, J.J.; Rosenberg, S.A.

    1985-01-01

    The authors previously reported that the adoptive transfer of lymphokine-activated killer (LAK) cells plus repetitive injections of recombinant interleukin 2 (IL 2) produced a marked reduction in established pulmonary metastases from a variety of murine sarcomas. The requirement for the exogenous administration of IL 2 prompted a subsequent examination of the role of IL 2 in the in vivo function of transferred LAK cells. The in vivo proliferation and migration patterns of lymphoid cells in C57BL/6 mice were examined after i.v. transfer of LAK cells alone, i.p. injection of IL 2 alone, or the combination of LAK cells and IL 2. A model for in vivo labeling of the DNA of dividing cells was used in which mice were injected with 5-[ 125 I]-iodo-2'-deoxyuridine ( 125 IUdR) and, 20 hr later, their tissues were removed and were counted in a gamma analyzer. A proliferation index (PI) was calculated by dividing the mean cpm of organs of experimentally treated mice by the mean cpm of organs of control mice. In animals given LAK cells alone, the lungs and liver demonstrated little if any uptake of 125 IUdR above saline-treated controls, whereas the same organs of mice receiving 6000 U of IL 2 alone displayed higher radiolabel incorporation. When mice were given LAK cells plus 6000 U of IL 2, their tissues showed an additional increase in 125 IUdR uptake

  5. Trichomonas vaginalis cathepsin D-like aspartic proteinase (Tv-CatD) is positively regulated by glucose and degrades human hemoglobin.

    Science.gov (United States)

    Mancilla-Olea, Maria Inocente; Ortega-López, Jaime; Figueroa-Angulo, Elisa E; Avila-González, Leticia; Cárdenas-Guerra, Rosa Elena; Miranda-Ozuna, Jesús F T; González-Robles, Arturo; Hernández-García, Mar Saraí; Sánchez-Ayala, Lizbeth; Arroyo, Rossana

    2018-04-01

    Trichomonas vaginalis genome encodes ∼440 proteases, six of which are aspartic proteases (APs). However, only one belongs to a clan AA (EC 3.4.23.5), family A1 (pepsin A), cathepsin D-like protease. This AP is encoded by an 1113-bp gene (tv-catd), which translates into a 370-aa residues zymogen of 40.7-kDa and a theoretical pI of 4.6, generating a ∼35 kDa active enzyme after maturation (Tv-CatD). The goal of this study was to identify and analyze the effect of glucose on the expression of Tv-CatD at the transcript and protein levels, subcellular localization, and proteolytic activity. The qRT-PCR assays showed a ∼2-fold increase in tv-catd mRNA under high-glucose (HG) conditions compared to glucose-restriction (GR) conditions. We amplified, cloned, and expressed the tv-catd gene, and purified the recombinant precursor enzyme (Tv-CatDr) to generate a polyclonal antibody (anti-Tv-CatDr). Western blot (WB) and immunolocalization assays showed that glucose increases the amount of Tv-CatD in different subcellular localizations and in in vitro secretions. Additionally, Tv-CatD proteolytic activity was detected in protease-resistant extracts (PREs) using a synthetic fluorogenic peptide specific for cathepsin D/E APs at different pHs and in the presence of AP inhibitors. In a two-dimensional (2-DE) WB analysis of a PRE from parasites grown under GR and HG conditions, an anti-Tv-CatDr antibody detected a 35-kDa protein spot at pI 5.0 identified as the mature Tv-CatD form by mass spectrometry that showed proteolytic activity in 2-DE zymograms copolymerized with hemoglobin under both glucose conditions. Thus, Tv-CatD could be involved in trichomonal hemolysis. Copyright © 2018 Elsevier Ltd. All rights reserved.

  6. Modulation of cathepsin G expression in severe atopic dermatitis following medium-dose UVA1 phototherapy

    OpenAIRE

    Breuckmann, Frank; von Kobyletzki, Gregor; Avermaete, Annelies; Kreuter, Alexander; Altmeyer, Peter; Gambichler, Thilo

    2002-01-01

    Abstract Background During the last decade, medium-dose UVA1 phototherapy (50 J/cm2) has achieved great value within the treatment of severe atopic dermatitis (AD). The purpose of our study was to investigate to what extent UVA1 irradiation is able to modulate the status of protease activity by the use of a monoclonal antibody labeling cathepsin G. Methods In order to further elucidate the mechanisms by which medium-dose UVA1 irradiation leads to an improvement of skin status in patients with...

  7. Extracellular cystatin SN and cathepsin B prevent cellular senescence by inhibiting abnormal glycogen accumulation.

    Science.gov (United States)

    Oh, Sang-Seok; Park, Soojong; Lee, Ki-Won; Madhi, Hamadi; Park, Sae Gwang; Lee, Hee Gu; Cho, Yong-Yeon; Yoo, Jiyun; Dong Kim, Kwang

    2017-04-06

    Cystatin SN (CST1), a known inhibitor of cathepsin B (CatB), has important roles in tumor development. Paradoxically, CatB is a member of the cysteine cathepsin family that acts in cellular processes, such as tumor development and invasion. However, the relationship between CST1 and CatB, and their roles in tumor development are poorly understood. In this study, we observed that the knockdown of CST1 induced the activity of senescence-associated β-galactosidase, a marker of cellular senescence, and expression of senescence-associated secretory phenotype genes, including interleukin-6 and chemokine (C-C motif) ligand 20, in MDA-MB-231 and SW480 cancer cells. Furthermore, CST1 knockdown decreased extracellular CatB activity, and direct CatB inhibition, using specific inhibitors or shCatB, induced cellular senescence. Reconstitution of CST1 restored CatB activity and inhibited cellular senescence in CST1 knockdown cells. CST1 knockdown or CatB inhibition increased glycogen synthase (GS) kinase 3β phosphorylation at serine 9, resulting in the activation of GS and the induction of glycogen accumulation associated with cellular senescence. Importantly, CST1 knockdown suppressed cancer cell proliferation, soft agar colony growth and tumor growth in a xenograft model. These results indicate that CST1-mediated extracellular CatB activity enhances tumor development by preventing cellular senescence. Our findings suggest that antagonists of CST1 or inhibitors of CatB are potential anticancer agents.

  8. Cathepsin B & L are not required for ebola virus replication.

    Science.gov (United States)

    Marzi, Andrea; Reinheckel, Thomas; Feldmann, Heinz

    2012-01-01

    Ebola virus (EBOV), family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV). EBOV encodes one viral surface glycoprotein (GP), which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL), which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus) was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control), catB(-/-) and catL(-/-) mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

  9. Cathepsin B & L are not required for ebola virus replication.

    Directory of Open Access Journals (Sweden)

    Andrea Marzi

    Full Text Available Ebola virus (EBOV, family Filoviridae, emerged in 1976 on the African continent. Since then it caused several outbreaks of viral hemorrhagic fever in humans with case fatality rates up to 90% and remains a serious Public Health concern and biothreat pathogen. The most pathogenic and best-studied species is Zaire ebolavirus (ZEBOV. EBOV encodes one viral surface glycoprotein (GP, which is essential for replication, a determinant of pathogenicity and an important immunogen. GP mediates viral entry through interaction with cellular surface molecules, which results in the uptake of virus particles via macropinocytosis. Later in this pathway endosomal acidification activates the cysteine proteases Cathepsin B and L (CatB, CatL, which have been shown to cleave ZEBOV-GP leading to subsequent exposure of the putative receptor-binding and fusion domain and productive infection. We studied the effect of CatB and CatL on in vitro and in vivo replication of EBOV. Similar to previous findings, our results show an effect of CatB, but not CatL, on ZEBOV entry into cultured cells. Interestingly, cell entry by other EBOV species (Bundibugyo, Côte d'Ivoire, Reston and Sudan ebolavirus was independent of CatB or CatL as was EBOV replication in general. To investigate whether CatB and CatL have a role in vivo during infection, we utilized the mouse model for ZEBOV. Wild-type (control, catB(-/- and catL(-/- mice were equally susceptible to lethal challenge with mouse-adapted ZEBOV with no difference in virus replication and time to death. In conclusion, our results show that CatB and CatL activity is not required for EBOV replication. Furthermore, EBOV glycoprotein cleavage seems to be mediated by an array of proteases making targeted therapeutic approaches difficult.

  10. Cathepsin G Controls Arterial But Not Venular Myeloid Cell Recruitment

    NARCIS (Netherlands)

    Ortega-Gomez, Almudena; Salvermoser, Melanie; Rossaint, Jan; Pick, Robert; Brauner, Janine; Lemnitzer, Patricia; Tilgner, Jessica; de Jong, Renske J.; Megens, Remco T. A.; Jamasbi, Janina; Döring, Yvonne; Pham, Christine T.; Scheiermann, Christoph; Siess, Wolfgang; Drechsler, Maik; Weber, Christian; Grommes, Jochen; Zarbock, Alexander; Walzog, Barbara; Soehnlein, Oliver

    2016-01-01

    Therapeutic targeting of arterial leukocyte recruitment in the context of atherosclerosis has been disappointing in clinical studies. Reasons for such failures include the lack of knowledge of arterial-specific recruitment patterns. Here we establish the importance of the cathepsin G (CatG) in the

  11. Structure of the periodontium in cathepsin C-deficient mice

    NARCIS (Netherlands)

    de Haar, Susanne F.; Tigchelaar-Gutter, Wikky; Everts, Vincent; Beertsen, Wouter

    2006-01-01

    Papillon-Lefevre syndrome is characterized by increased susceptibility to early-onset periodontitis and is caused by mutations in the cathepsin C gene. How deficiency of the enzyme relates to an increased periodontal infection risk is still not entirely clear. One possibility is that the deficiency

  12. Cleavage of desmin by cysteine proteases: Calpains and cathepsin B

    DEFF Research Database (Denmark)

    Baron, Caroline; Jacobsen, S.; Purslow, P.P.

    2004-01-01

    The intermediate filament protein, desmin, was purified from pork longissimus dorsi and incubated with either P-calpain, m-calpain or cathepsin B. Proteolysis of desmin was followed using SDS-PAGE and Western blotting. After incubation of desmin with the proteases, cleavage sites on the desmin mo...

  13. Endogenous acute phase serum amyloid A lacks pro-inflammatory activity, contrasting the two recombinant variants that activate human neutrophils through different receptors

    Directory of Open Access Journals (Sweden)

    Karin eChristenson

    2013-04-01

    Full Text Available Most notable among the acute phase proteins is serum amyloid A (SAA, levels of which can increase 1000-fold during infections, aseptic inflammation, and/or trauma. Chronically elevated SAA levels are associated with a wide variety of pathological conditions, including obesity and rheumatic diseases. Using a recombinant hybrid of the two human SAA isoforms (SAA1 and 2 that does not exist in vivo, numerous in vitro studies have given rise to the notion that acute phase SAA is a pro-inflammatory molecule with cytokine-like properties. It is however unclear whether endogenous acute phase SAA per se mediates pro-inflammatory effects. We tested this in samples from patients with inflammatory arthritis and in a transgenic mouse model that expresses human SAA1. Endogenous human SAA did not drive production of pro-inflammatory IL-8/KC in either of these settings. Human neutrophils derived from arthritis patients displayed no signs of activation, despite being exposed to severely elevated SAA levels in circulation, and SAA-rich sera also failed to activate cells in vitro. In contrast, two recombinant SAA variants (the hybrid SAA and SAA1 both activated human neutrophils, inducing L-selectin shedding, production of reactive oxygen species, and production of IL-8. The hybrid SAA was approximately 100-fold more potent than recombinant SAA1. Recombinant hybrid SAA and SAA1 activated neutrophils through different receptors, with recombinant SAA1 being a ligand for formyl peptide receptor 2 (FPR2. We conclude that even though recombinant SAAs can be valuable tools for studying neutrophil activation, they do not reflect the nature of the endogenous protein.

  14. Crystallographic, DFT and docking (cathepsin B) studies on an organotellurium(IV) compound

    International Nuclear Information System (INIS)

    Caracelli, Ignez; Maganhi, Stella H.; Zukerman-Schpector, Julio; Sousa Madureira, Lucas; Stefani, Helio A.; Guadagnin, Rafael C.; Tiekink, Edward R.T.

    2016-01-01

    Some biologically active organotellurium compounds exhibit inhibitory potency against cathepsin B. In this study, an alkyl derivative, viz. [CH 3 (CH 2 ) 2 C(I)=C(H)](nBu)TeI 2 , 1, has been structurally characterised by X-ray crystallography and shown to be coordinated within a C 2 I 2 donor set. When the stereochemically active lone pair of electrons is taken into account, a distorted trigonal bipyramidal geometry results with the iodide atoms in axial positions. Both intra- and inter-molecular Te..I interactions are also noted. If all interactions are considered, the coordination geometry is based on a Ψ-pentagonal bipyramidal geometry. An unusual feature of the structure is the curving of the functionalised C 5 chain. This feature has been explored by DFT methods and shown to arise as a result of close C-H..I interactions. A docking study (cathepsin B) was performed to understand the inhibition mechanism and to compare the new results with previous observations. Notably, 1 has the same pose exhibited by analogous biologically active compounds with aryl groups. Thus, the present study suggests that (alkyl) 2 TeX 2 compounds should also be evaluated for biological activity.

  15. Crystallographic, DFT and docking (cathepsin B) studies on an organotellurium(IV) compound

    Energy Technology Data Exchange (ETDEWEB)

    Caracelli, Ignez; Maganhi, Stella H. [Univ. Federal de Sao Carlos (Brazil). BioMat; Zukerman-Schpector, Julio; Sousa Madureira, Lucas [Univ. Federal de Sao Carlos (Brazil). Lab. de Cristalografia, Estereodinamica e Modelagem Molecular; Stefani, Helio A. [Sao Paulo Univ. (Brazil). Dept. de Farmacia; Guadagnin, Rafael C. [Univ. Federal de Sao Paulo, Diadema (Brazil). Inst. e Ciencias Mabientais, Quimicas e Farmaceuticas; Tiekink, Edward R.T. [Sunway Univ., Selangor Darul Ehsan (Malaysia). Centre for Crystalline Materials

    2016-08-01

    Some biologically active organotellurium compounds exhibit inhibitory potency against cathepsin B. In this study, an alkyl derivative, viz. [CH{sub 3}(CH{sub 2}){sub 2}C(I)=C(H)](nBu)TeI{sub 2}, 1, has been structurally characterised by X-ray crystallography and shown to be coordinated within a C{sub 2}I{sub 2} donor set. When the stereochemically active lone pair of electrons is taken into account, a distorted trigonal bipyramidal geometry results with the iodide atoms in axial positions. Both intra- and inter-molecular Te..I interactions are also noted. If all interactions are considered, the coordination geometry is based on a Ψ-pentagonal bipyramidal geometry. An unusual feature of the structure is the curving of the functionalised C{sub 5} chain. This feature has been explored by DFT methods and shown to arise as a result of close C-H..I interactions. A docking study (cathepsin B) was performed to understand the inhibition mechanism and to compare the new results with previous observations. Notably, 1 has the same pose exhibited by analogous biologically active compounds with aryl groups. Thus, the present study suggests that (alkyl){sub 2}TeX{sub 2} compounds should also be evaluated for biological activity.

  16. Expression and characterization of recombinant single-chain salmon class I MHC fused with beta2-microglobulin with biological activity

    DEFF Research Database (Denmark)

    Zhao, Heng; Stet, René J M; Skjødt, Karsten

    2008-01-01

    Heterodimeric class I major histocompatibility complex (MHC) molecules consist of a putative 45-kDa heavy chain and a 12-kDa beta2-microglobulin (beta2m) light chain. The knowledge about MHC genes in Atlantic salmon accumulated during the last decade has allowed us to generate soluble and stable ...... MHC class I molecules with biological activity. We report here the use of a bacterial expression system to produce the recombinant single-chain MHC molecules based on a specific allele Sasa-UBA*0301. This particular allele was selected because previous work has shown its association...... antibodies were successfully produced against both the MHC class I heavy chain and beta(2)m, and showed binding to the recombinant molecule. The recombinant complex Sasabeta2mUBA*0301 was expressed and isolated; the production was scaled up by adjusting to its optimal conditions. Subsequently......, the recombinant proteins were purified by affinity chromatography using mAb against beta2m and alpha3. Eluates were analyzed by Western blot and refolded by the removal of denaturant. The correct folding was confirmed by measuring its binding capacity against mAb produced to recognize the native form of MHC...

  17. Recombinant Programming

    OpenAIRE

    Pawlak , Renaud; Cuesta , Carlos; Younessi , Houman

    2004-01-01

    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. Potency of full-length MGF to induce maximal activation of the IGF-I R Is similar to recombinant human IGF-I at high equimolar concentrations

    NARCIS (Netherlands)

    J.A.M.J.L. Janssen (Joseph); L.J. Hofland (Leo); C.J. Strasburger; E.S.R.D. Van Dungen (Elisabeth S.R. Den); M. Thevis (Mario)

    2016-01-01

    textabstractAims To compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin

  19. Intravenous recombinant tissue plasminogen activator for acute ischemic stroke: a feasibility and safety study

    Directory of Open Access Journals (Sweden)

    Sadeghi-Hokmabadi E

    2016-10-01

    Full Text Available Elyar Sadeghi-Hokmabadi, Mehdi Farhoudi, Aliakbar Taheraghdam, Mazyar Hashemilar, Daryous Savadi-Osguei, Reza Rikhtegar, Kaveh Mehrvar, Ehsan Sharifipour, Parisa Youhanaee, Reshad Mirnour Neurosciences Research Center, Neurology Department, Tabriz University of Medical Sciences, Tabriz, East Azerbaijan, Iran Background: In developing countries, intravenous thrombolysis (IVT is available at a limited number of centers. This study aimed to assess the feasibility and safety of IVT at Tabriz Imam Reza Hospital. Methods: In a prospective study, over a 55-month period, any patient at the hospital for whom stroke code had been activated was enrolled in the study. Data on demographic characteristics, stroke risk factors, admission blood pressure, blood tests, findings of brain computed tomography (CT scans, time of symtom onset, time of arrival to the emergency department, time of stroke code activation, time of CT scan examination, and the time of recombinant tissue plasminogen activator administration were recorded. National Institutes of Health Stroke Scale assessments were performed before IVT bolus, at 36 hours, at either 7 days or discharge (which ever one was earlier, and at 3-month follow-up. Brain CT scans were done for all patients before and 24 hours after the treatment. Results: Stroke code was activated for 407 patients and IVT was done in 168 patients. The rate of functional independence (modified Rankin Scale [mRS] 0–1 at 3 months was 39.2% (62/158. The mortality rate at day 7 was 6% (10/168. Hemorrhagic transformation was noted in 16 patients (9.5%. Symptomatic intracranial hemorrhage occurred in 5 (3%, all of which were fatal. One case of severe urinary bleeding and one other fatal case of severe angioedema were observed. Conclusion: During the first 4–5 years of administration of IVT in the hospital, it was found to be feasible and safe, but to increase the efficacy, poststroke care should be more organized and a stroke center

  20. Effective DNA Inhibitors of Cathepsin G by In Vitro Selection

    Science.gov (United States)

    Gatto, Barbara; Vianini, Elena; Lucatello, Lorena; Sissi, Claudia; Moltrasio, Danilo; Pescador, Rodolfo; Porta, Roberto; Palumbo, Manlio

    2008-01-01

    Cathepsin G (CatG) is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions. PMID:19325843

  1. Effective DNA Inhibitors of Cathepsin G by In Vitro Selection

    Directory of Open Access Journals (Sweden)

    Manlio Palumbo

    2008-06-01

    Full Text Available Cathepsin G (CatG is a chymotrypsin-like protease released upon degranulation of neutrophils. In several inflammatory and ischaemic diseases the impaired balance between CatG and its physiological inhibitors leads to tissue destruction and platelet aggregation. Inhibitors of CatG are suitable for the treatment of inflammatory diseases and procoagulant conditions. DNA released upon the death of neutrophils at injury sites binds CatG. Moreover, short DNA fragments are more inhibitory than genomic DNA. Defibrotide, a single stranded polydeoxyribonucleotide with antithrombotic effect is also a potent CatG inhibitor. Given the above experimental evidences we employed a selection protocol to assess whether DNA inhibition of CatG may be ascribed to specific sequences present in defibrotide DNA. A Selex protocol was applied to identify the single-stranded DNA sequences exhibiting the highest affinity for CatG, the diversity of a combinatorial pool of oligodeoxyribonucleotides being a good representation of the complexity found in defibrotide. Biophysical and biochemical studies confirmed that the selected sequences bind tightly to the target enzyme and also efficiently inhibit its catalytic activity. Sequence analysis carried out to unveil a motif responsible for CatG recognition showed a recurrence of alternating TG repeats in the selected CatG binders, adopting an extended conformation that grants maximal interaction with the highly charged protein surface. This unprecedented finding is validated by our results showing high affinity and inhibition of CatG by specific DNA sequences of variable length designed to maximally reduce pairing/folding interactions.

  2. Preparation of thermosensitive magnetic liposome encapsulated recombinant tissue plasminogen activator for targeted thrombolysis

    Energy Technology Data Exchange (ETDEWEB)

    Hsu, Hao-Lung [Department of Chemical and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan, ROC (China); Chen, Jyh-Ping, E-mail: jpchen@mail.cgu.edu.tw [Department of Chemical and Materials Engineering, Chang Gung University, Kwei-San, Taoyuan 33302, Taiwan, ROC (China); Department of Plastic and Reconstructive Surgery and Craniofacial Research Center, Chang Gung Memorial Hospital, Kwei-San, Taoyuan 33305, Taiwan, ROC (China); Graduate Institute of Health Industry and Technology, Research Center for Industry of Human Ecology, Chang Gung University of Science and Technology, Kwei-San, Taoyuan 33302, Taiwan, ROC (China); Department of Materials Engineering, Ming Chi University of Technology, Tai-Shan, New Taipei City 24301, Taiwan, ROC (China)

    2017-04-01

    Recombinant tissue plasminogen activator (rtPA) was encapsulated in thermosensitive magnetic liposome (TML) prepared from 1,2-dipalmitoyl-sn-glycero-3-phosphocholine, distearolyphosphatidyl ethanolamine-N-poly(ethylene glycol) 2000, cholesterol and Fe{sub 3}O{sub 4} magnetic nanoparticles by solvent evaporation/sonication and freeze-thaw cycles method. Response surface methodology was proved to be a powerful tool to predict the drug encapsulation efficiency and temperature-sensitive drug release. Validation experiments verified the accuracy of the model that provides a simple and effective method for fabricating TML with controllable encapsulation efficiency and predictable temperature-sensitive drug release behavior. The prepared samples were characterized for physico-chemical properties by dynamic light scattering, transmission electron microscopy, X-ray diffraction and differential scanning calorimetry. Temperature-sensitive release of rtPA could be confirmed from in vitro thrombolysis experiments. A thrombolytic drug delivery system using TML could be proposed for magnetic targeted delivery of rtPA to the site of thrombus followed by temperature-triggered controlled drug release in an alternating magnetic field. - Highlights: • rtPA and Fe{sub 3}O{sub 4} MNP were encapsulated in thermosensitive magnetic liposome (TML). • RSM could predict the drug encapsulation efficiency and temperature-sensitive drug release from TML. • Temperature-sensitive release of rtPA was confirmed from in vitro thrombolysis experiments. • TML-rtPA will be useful as a magnetic targeted nanodrug to improve clinical thrombolytic therapy.

  3. Novel recombinant human lactoferrin: differential activation of oxidative stress related gene expression.

    Science.gov (United States)

    Kruzel, Marian L; Actor, Jeffrey K; Zimecki, Michał; Wise, Jasen; Płoszaj, Paulina; Mirza, Shaper; Kruzel, Mark; Hwang, Shen-An; Ba, Xueqing; Boldogh, Istvan

    2013-12-01

    Lactoferrin, an iron-binding protein found in high concentrations in mammalian exocrine secretions, is an important component of the host defense system. It is also a major protein of the secondary granules of neutrophils from which is released upon activation. Due to its potential clinical utility, recombinant human lactoferrin (rhLF) has been produced in various eukaryotic expression systems; however, none of these are fully compatible with humans. Most of the biopharmaceuticals approved by the FDA for use in humans are produced in mammalian expression systems. The Chinese hamster ovary cells (CHO) have become the system of choice for proteins that require post-translational modifications, such as glycoproteins. The aim of this study was to scale-up expression and purification of rhLF in a CHO expression system, verify its glycan primary structure, and assess its biological properties in cell culture models. A stable CHO cell line producing >200mg/L of rhLF was developed and established. rhLF was purified by a single-step cation-exchange chromatography procedure. The highly homogenous rhLF has a molecular weight of approximately 80 kDa. MALDI-TOF mass spectrometric analysis revealed N-linked, partially sialylated glycans at two glycosylation sites, typical for human milk LF. This novel rhLF showed a protective effect against oxidative stress in a similar manner to its natural counterpart. In addition, rhLF revealed a modulatory effect on cellular redox via upregulation of key antioxidant enzymes. These data imply that the CHO-derived rhLF is fully compatible with the native molecule, thus it has promise for human therapeutic applications. Copyright © 2013 Elsevier B.V. All rights reserved.

  4. The Effect of Recombinant Activated Factor VII on Mortality in Combat-Related Casualties With Severe Trauma and Massive Transfusion

    Science.gov (United States)

    2008-02-01

    acidosis and hypocalcemia are corrected. In addition, these guidelines recommend rFVIIa for patients with adequate concentrations of platelets, fibrinogen...1.00 Data on physiologic cause of death available for 51 of 53 deaths recorded. rFVIIa, recombinant activated factor VII; CNS, central nervous ... system ; MOF, multi-organ failure. Table 10 Comparison of Adverse Events Between Study Groups Variable rFVIIa N 75 rFVIIa N 49 p Value Bacteremia 12

  5. Development of an SPR imaging biosensor for determination of cathepsin G in saliva and white blood cells

    International Nuclear Information System (INIS)

    Gorodkiewicz, E.; Wojtulewski, K.; Regulska, E.

    2011-01-01

    Cathepsin G (CatG) is an endopeptidase that is associated with the early immune response. The synthetic compound cathepsin G inhibitor I (CGI-I) was tested for its ability to inhibit the activity of CatG via a new surface plasmon resonance imaging assay. CGI-I was immobilized on the gold surface of an SPR sensor that was first modified with 1-octadecanethiol. A concentration of CGI-I equal to 4.0 μg.mL -1 and a pH of 8.0 were found to give the best results. The dynamic response of the sensor ranges from 0. 25 to 1. 5 ng.mL -1 , and the detection limit is 0. 12 ng.mL -1 . The sensor was applied to detect CatG in human saliva and white blood cells. (author)

  6. Purification and characterization of cathepsin L in arrowtooth flounder (Atheresthes stomias) muscle.

    Science.gov (United States)

    Visessanguan, Wonnop; Benjakul, Soottawat; An, Haejung

    2003-03-01

    A predominant, heat-activated proteinase in muscle extract of arrowtooth flounder (Atheresthes stomias) was purified to 55-fold by heat treatment, followed by a series of chromatographic separations. The apparent molecular mass of the purified enzyme was 27 kDa by size exclusion chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteinase had high affinity and activity toward Z-Phe-Arg-NMec with K(m) and k(cat) values of 8.2 microM and 12.2/s, respectively. Activity was inhibited by sulfhydryl reagents and activated by reducing agents. The purified proteinase displayed optimal activity at pH 5.0-5.5 and 60 degrees C, respectively. Consistent with the properties of proteases from other species, the heat-activated proteinase in arrowtooth flounder can be identified as cathepsin L.

  7. β-Endorphin biotransformation in brain: Formation of γ-endorphin by a synaptosomal plasma membrane associated endopeptidase distinct from cathepsin D

    NARCIS (Netherlands)

    Burbach, J.P.H.; Loeber, J.G.; Verhoef, J.; Kloet, E.R. de

    1980-01-01

    cSPM preparations of rat brain contain a peptidase activity which generates γ-endorphin from β-endorphin. Some properties of this enzyme were studied and compared with those of cathepsin D. Maximal accumulation of γ-endorphin upon digestion of β-endorphin with a cSPM preparation was found at neutral

  8. Evaluation of recombinant activated protein C for severe sepsis at a tertiary academic medical center

    Directory of Open Access Journals (Sweden)

    Anger KE

    2013-06-01

    Full Text Available Kevin E Anger,1 Jeremy R DeGrado,1 Bonnie C Greenwood,1 Steven A Cohen,2 Paul M Szumita1 1Department of Pharmacy, Brigham and Women’s Hospital, Boston, MA, USA; 2Department of Family Medicine and Population Health, Division of Epidemiology, Virginia Commonwealth University, Richmond, VA, USA Purpose: Early clinical trials of recombinant human activated protein C (rhAPC for severe sepsis excluded patients at high risk of bleeding. Recent literature suggests bleeding rates are higher in clinical practice and may be associated with worsened outcomes. Our objective was to evaluate baseline demographics; incidence, and risk factors for major bleeding; and mortality of patients receiving rhAPC for severe sepsis at our institution. Methods: A retrospective study was performed for all patients receiving rhAPC for treatment of severe sepsis at a tertiary academic medical center from January 2002 to June 2009. Demographic information, clinical variables, intensive care unit, and hospital outcomes were recorded. Results: Of the 156 patients that received rhAPC, 54 (34.6% did not meet institutional criteria for safe use at baseline due to bleeding precaution or contraindication. Twenty-three (14.7% patients experienced a major bleeding event. Multivariate analysis demonstrated baseline International Normalized Ratio ≥2.5 (odds ratio [OR] 3.68, 95% confidence interval [CI]: 1.28–10.56; P = 0.03 and platelet count ≤100 × 103/mm3 (OR 2.86, 95% CI: 1.07–7.67; P = 0.01 as significant predictors of a major bleed. Overall hospital mortality was 57.7%. Multivariate analysis demonstrated the presence of ≥3 organ dysfunctions (OR 2.46, 95% CI: 1.19–5.09; P < 0.05 and medical intensive care unit admission (OR 1.99, 95% CI: 1.00–3.98; P = 0.05 were independent variables associated with hospital mortality. Conclusion: Patients receiving rhAPC at our institution had higher APACHE II scores, mortality, and major bleeding events than published

  9. Expression of active recombinant human alpha 1-antitrypsin in transgenic rabbits

    NARCIS (Netherlands)

    Massoud, M.; Bischoff, Rainer; Dalemans, W.; Pointu, H.; Attal, J.; Schultz, H.; Clesse, D.; Stinnakre, M.G.; Pavirani, A.; Houdebine, L.M.

    1991-01-01

    A DNA construct containing the human alpha 1-antitrypsin gene including 1.5 and 4 kb of 5' and 3' flanking sequences, was microinjected into the pronucleus of rabbit embryos. The recombinant human protein was (a) expressed in the blood circulation of F0 and F1 transgenic rabbits at an average

  10. Influence of cardiopulmonary bypass on the interaction of recombinant factor VIIa with activated platelets

    DEFF Research Database (Denmark)

    Kjalke, M.; Runge, M.; Rojkjaer, R.

    2009-01-01

    Recombinant factor VIIa (rFVIIa) interacts preferentially with coated platelets characterized by a high exposure of phosphatidyl serine (PS), FV, FVIII, FIX, and FX binding, and fibrinogen. Cardiopulmonary bypass (CPB) is known to impair platelet function. In this study, the influence of CPB...

  11. Intraoperative use of low-dose recombinant activated factor VII during thoracic aortic operations.

    Science.gov (United States)

    Andersen, Nicholas D; Bhattacharya, Syamal D; Williams, Judson B; Fosbol, Emil L; Lockhart, Evelyn L; Patel, Mayur B; Gaca, Jeffrey G; Welsby, Ian J; Hughes, G Chad

    2012-06-01

    Numerous studies have supported the effectiveness of recombinant activated factor VII (rFVIIa) for the control of bleeding after cardiac procedures; however safety concerns persist. Here we report the novel use of intraoperative low-dose rFVIIa in thoracic aortic operations, a strategy intended to improve safety by minimizing rFVIIa exposure. Between July 2005 and December 2010, 425 consecutive patients at a single referral center underwent thoracic aortic operations with cardiopulmonary bypass (CPB); 77 of these patients received intraoperative low-dose rFVIIa (≤60 μg/kg) for severe coagulopathy after CPB. Propensity matching produced a cohort of 88 patients (44 received intraoperative low-dose rFVIIa and 44 controls) for comparison. Matched patients receiving intraoperative low-dose rFVIIa got an initial median dose of 32 μg/kg (interquartile range [IQR], 16-43 μg/kg) rFVIIa given 51 minutes (42-67 minutes) after separation from CPB. Patients receiving intraoperative low-dose rFVIIa demonstrated improved postoperative coagulation measurements (partial thromboplastin time 28.6 versus 31.5 seconds; p=0.05; international normalized ratio, 0.8 versus 1.2; pproduct transfusions (2.5 versus 5.0 units; p=0.05) compared with control patients. No patient receiving intraoperative low-dose rFVIIa required postoperative rFVIIa administration or reexploration for bleeding. Rates of stroke, thromboembolism, myocardial infarction, and other adverse events were equivalent between groups. Intraoperative low-dose rFVIIa led to improved postoperative hemostasis with no apparent increase in adverse events. Intraoperative rFVIIa administration in appropriately selected patients may correct coagulopathy early in the course of refractory blood loss and lead to improved safety through the use of smaller rFVIIa doses. Appropriately powered randomized studies are necessary to confirm the safety and efficacy of this approach. Copyright © 2012 The Society of Thoracic Surgeons

  12. Plasmid construction using recombination activity in the fission yeast Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ayako Chino

    Full Text Available BACKGROUND: Construction of plasmids is crucial in modern genetic manipulation. As of now, the common method for constructing plasmids is to digest specific DNA sequences with restriction enzymes and to ligate the resulting DNA fragments with DNA ligase. Another potent method to construct plasmids, known as gap-repair cloning (GRC, is commonly used in the budding yeast Saccharomyces cerevisiae. GRC makes use of the homologous recombination activity that occurs within the yeast cells. Due to its flexible design and efficiency, GRC has been frequently used for constructing plasmids with complex structures as well as genome-wide plasmid collections. Although there have been reports indicating GRC feasibility in the fission yeast Schizosaccharomyces pombe, this species is not commonly used for GRC as systematic studies of reporting GRC efficiency in S. pombe have not been performed till date. METHODOLOGY/PRINCIPAL FINDINGS: We investigated GRC efficiency in S. pombe in this study. We first showed that GRC was feasible in S. pombe by constructing a plasmid that contained the LEU2 auxotrophic marker gene in vivo and showed sufficient efficiency with short homology sequences (>25 bp. No preference was shown for the sequence length from the cut site in the vector plasmid. We next showed that plasmids could be constructed in a proper way using 3 DNA fragments with 70% efficiency without any specific selections being made. The GRC efficiency with 3 DNA fragments was dramatically increased >95% in lig4Delta mutant cell, where non-homologous end joining is deficient. Following this approach, we successfully constructed plasmid vectors with leu1+, ade6+, his5+, and lys1+ markers with the low-copy stable plasmid pDblet as a backbone by applying GRC in S. pombe. CONCLUSIONS/SIGNIFICANCE: We concluded that GRC was sufficiently feasible in S. pombe for genome-wide gene functional analysis as well as for regular plasmid construction. Plasmids with different

  13. [Clinical characteristics of human recombination activating gene 1 mutations in 8 immunodeficiency patients with diverse phenotypes].

    Science.gov (United States)

    Yu, G; Wang, W J; Liu, D R; Tao, Z F; Hui, X Y; Hou, J; Sun, J Q; Wang, X C

    2018-03-02

    Objective: To investigate the clinical characteristics of 8 immunodeficiency cases caused by human recombination activating gene 1 (RAG1) mutations, and to explore the relationship among genotypes, clinical manifestations and immunophenotypes. Methods: Clinical data were collected and analyzed from patients with RAG1 mutations who visited the Department of Clinical Immunology, Children's Hospital of Fudan University between October 2013 and June 2017. The data included clinical manifestations, immunophenotypes and genotypes. Results: A total of 8 patients were diagnosed with RAG1 deficiency (6 boys and 2 girls). The minimum age of onset was 2 months, and the maximum age was 4 months. The minimum age of diagnosis was 2 months, and the maximum age was 13 years. Four patients had a family history of infant death due to severe infections. Two cases were born to the same consanguineous parents. All cases had recurrent infections, including involvement of respiratory tract (8 cases), digestive tract (6 cases), urinary tract (1 case), and central nervous system (1 case). The pathogens of infection included bacteria, viruses and fungi. Rotavirus was found in 3 cases, cytomegalovirus (CMV) in 5 cases, bacillus Calmette-Guérin adverse reaction in 2 cases (1 of whom had a positive acid-fast smear from lymph node puncture fluid), fungal infection in 3 cases. One case had multiple nodular space-occupying lesions in lungs and abdominal cavity complicated with multiple bone destruction. The peripheral blood lymphocyte counts of all patients ranged between 0.1 ×10(9)/L and 3.3×10(9)/L (median, 0.65×10(9)/L). Eosinophilia was found in 3 cases (range, (0.48-1.69) ×10(9)/L). The patients were classified according to immunophenotype as severe combined immunodeficiency phenotype (4 cases), leaky severe combined immunodeficiency (2 cases), Omenn syndrome (1 case) and combined immunodeficiency (1 case) . Decreased serum IgG levels were found in 3 cases, increased serum IgM levels in

  14. Prophylactic administration of recombinant activated factor VII in coronary revascularization surgery

    Directory of Open Access Journals (Sweden)

    Mohamed Essam Abdel-Meguid

    2013-01-01

    Full Text Available Objective: The objective of this clinical trial is to study the effectiveness of administering recombinant activated factor VII (rFVIIa in reducing the amount of bleeding and the need for homologous blood and products transfusion in cardiac surgical coronary revascularization procedures done under cardiopulmonary bypass (CPB. Methods: In a randomized controlled prospective observational study, 30 patients were scheduled for elective cardiac revascularization under CPB. Patients were randomly allocated into two groups. In Group I (Control group, no rFVIIa was administered following CPB. In Group II (Study group, a dose of 90 ug/Kg of rFVIIa was administered following weaning off CPB. The total amount of chest tube drain during the 1 st 24 h following surgery was recorded as well as the qualitative and quantitative assessments of homologous blood and products transfusion. Serial analysis of hematological parameters including hemoglobin level and coagulation test in a definite data points was done. T0=baseline readings prior to CPB, T1=off CPB after protamine administration and before administration of the study drug, T2=on Cardiac Intensive Care Unit (CICU admission, T3=12 h post-CICU admission, and T4=24 h post-CICU admission. Results: Considering the total chest tube drainage, mean values showed statistically significant results with a P value of 0.001. Homologous blood and products transfusion were statistically lower in the study group. Regarding the mean values for hematological assessment, results showed statistically lower International Normalized Ratio values at CICU admission and 12 h post-CICU admission with a P value of 0.018 and 0.004, respectively. Also, the Partial Thromboplastin Time mean values were statistically lower at same timings with estimated P values of 0.04 and 0.001, respectively. Conclusion: It is concluded that the prophylactic use of rFVIIa in patients undergoing coronary revascularization surgery under the management

  15. Crystallization and preliminary crystallographic analysis of latent, active and recombinantly expressed aurone synthase, a polyphenol oxidase, from Coreopsis grandiflora

    Energy Technology Data Exchange (ETDEWEB)

    Molitor, Christian; Mauracher, Stephan Gerhard; Rompel, Annette, E-mail: annette.rompel@univie.ac.at [Universität Wien, Althanstrasse 14, 1090 Wien (Austria)

    2015-05-22

    Latent and active aurone synthase purified from petals of C. grandiflora (cgAUS1) were crystallized. The crystal quality of recombinantly expressed latent cgAUS1 was significantly improved by co-crystallization with the polyoxotungstate Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase-separation zone. Aurone synthase (AUS), a member of a novel group of plant polyphenol oxidases (PPOs), catalyzes the oxidative conversion of chalcones to aurones. Two active cgAUS1 (41.6 kDa) forms that differed in the level of phosphorylation or sulfation as well as the latent precursor form (58.9 kDa) were purified from the petals of Coreopsis grandiflora. The differing active cgAUS1 forms and the latent cgAUS1 as well as recombinantly expressed latent cgAUS1 were crystallized, resulting in six different crystal forms. The active forms crystallized in space groups P2{sub 1}2{sub 1}2{sub 1} and P12{sub 1}1 and diffracted to ∼1.65 Å resolution. Co-crystallization of active cgAUS1 with 1,4-resorcinol led to crystals belonging to space group P3{sub 1}21. The crystals of latent cgAUS1 belonged to space group P12{sub 1}1 and diffracted to 2.50 Å resolution. Co-crystallization of recombinantly expressed pro-AUS with the hexatungstotellurate(VI) salt Na{sub 6}[TeW{sub 6}O{sub 24}] within the liquid–liquid phase separation zone significantly improved the quality of the crystals compared with crystals obtained without hexatungstotellurate(VI)

  16. Efficient inhibition of cathepsin B by a secreted type 1 cystatin of Fasciola gigantica.

    Science.gov (United States)

    Siricoon, Sinee; Grams, Suksiri Vichasri; Grams, Rudi

    2012-12-01

    Cysteine proteases are important antigens in the liver fluke genus Fasciola, essential for infection, protection and nutrition. While their biochemistry, biological roles and application as vaccines have been thoroughly studied there is a lack of data concerning their regulation. In the present study we have continued our investigation of cysteine protease inhibitors in Fasciola gigantica and demonstrate, in comparison with FgStefin-1 and human cystatin C, that a second type 1 cystatin of the parasite, FgStefin-2, has been evolutionary adapted to block cathepsin B. The protein, which unusually for a type 1 cystatin carries a signal peptide, is expressed from the metacercarial to adult stage and located in the epithelial cells of the intestinal tract in all stages and in the prostate gland cells in adults. Both cell types may contribute to the released FgStefin-2 observed in the ES product of the parasite. Distinct isoforms of cathepsin B are essential for host tissue penetration during the early infection process and FgStefin-2 may act as key regulator, required to protect the minute juvenile from autoproteolysis. Expression in the prostate gland in the adult stage suggests an additional regulative role of cysteine protease activity in the reproductive system. Copyright © 2012 Elsevier B.V. All rights reserved.

  17. Cathepsin D and Its Prognostic Value in Neuroepithelial Brain Tumors

    OpenAIRE

    Pigac, Biserka; Dmitrović, Branko; Marić, Svjetlana; Mašić, Silvija

    2012-01-01

    Expression of Cathepsin D (Cath D) in some primary neuroepithelial brain tumors and its prognostic value were studied. The research included 65 samples of human primary neuroepithelial brain tumors. There were 50 glial tumors (10 diffuse astrocytomas (DA), 15 anaplastic astrocytomas (AA), 25 glioblastomas (GB), 15 embryonic tumors (15 medulloblastomas (MB) as well as 5 samples of normal brain tissue. Immunohistochemical method was applied to monitor diffuse positive reaction in the cytoplasm ...

  18. Structure of a Kunitz-type potato cathepsin D inhibitor

    Czech Academy of Sciences Publication Activity Database

    Guo, J.; Erskine, P. T.; Coker, A. R.; Wood, S. P.; Cooper, J. B.; Mareš, Michael; Baudyš, Miroslav

    2015-01-01

    Roč. 192, č. 3 (2015), s. 554-560 ISSN 1047-8477 R&D Projects: GA ČR GA15-18929S; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : potato cathepsin D inhibitor * Kunitz-type protease inhibitor * protein X-ray structure * reactive-site loop * docking Subject RIV: CE - Biochemistry Impact factor: 2.570, year: 2015

  19. Cathepsin E deficiency impairs autophagic proteolysis in macrophages.

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    Takayuki Tsukuba

    Full Text Available Cathepsin E is an endosomal aspartic proteinase that is predominantly expressed in immune-related cells. Recently, we showed that macrophages derived from cathepsin E-deficient (CatE(-/- mice display accumulation of lysosomal membrane proteins and abnormal membrane trafficking. In this study, we demonstrated that CatE(-/- macrophages exhibit abnormalities in autophagy, a bulk degradation system for aggregated proteins and damaged organelles. CatE(-/- macrophages showed increased accumulation of autophagy marker proteins such as LC3 and p62, and polyubiquitinated proteins. Cathepsin E deficiency also altered autophagy-related signaling pathways such as those mediated by the mammalian target of rapamycin (mTOR, Akt, and extracellular signal-related kinase (ERK. Furthermore, immunofluorescence microscopy analyses showed that LC3-positive vesicles were merged with acidic compartments in wild-type macrophages, but not in CatE(-/- macrophages, indicating inhibition of fusion of autophagosome with lysosomes in CatE(-/- cells. Delayed degradation of LC3 protein was also observed under starvation-induced conditions. Since the autophagy system is involved in the degradation of damaged mitochondria, we examined the accumulation of damaged mitochondria in CatE(-/- macrophages. Several mitochondrial abnormalities such as decreased intracellular ATP levels, depolarized mitochondrial membrane potential, and decreased mitochondrial oxygen consumption were observed. Such mitochondrial dysfunction likely led to the accompanying oxidative stress. In fact, CatE(-/- macrophages showed increased reactive oxygen species (ROS production and up-regulation of oxidized peroxiredoxin-6, but decreased antioxidant glutathione. These results indicate that cathepsin E deficiency causes autophagy impairment concomitantly with increased aberrant mitochondria as well as increased oxidative stress.

  20. Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect.

    Science.gov (United States)

    Mathijssen, Natascha C J; Masereeuw, Rosalinde; Holme, Pal Andre; van Kraaij, Marian G J; Laros-van Gorkom, Britta A P; Peyvandi, Flora; van Heerde, Waander L

    2013-08-01

    Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. Ten factor VII deficient patients were treated with either recombinant activated (20 μg/kg) or plasma-derived (25 IU/kg) factor VII in a cross-over design. Pharmacokinetic parameters were analyzed through activated factor VII activity, factor VII clotting activity, and factor VII antigen levels on depicted time points. Factor VII activity half-lifes, determined by non-compartmental and one-compartmental analysis (results in brackets), were shorter for recombinant activated (1.4h; 0.7h) than for plasma-derived factor VII (6.8h; 3.2h); both recombinant activated (5.1h; 2.1h and plasma-derived factor VII (5.8h; 3.2h) resulted in longer half-lives of factor VII antigen. Activated factor VII half-lives (based on activated factor VII activity levels) were significantly higher compared to factor VII clotting activity (1.6h; 0.9h). Volumes of distribution were significantly higher for activated factor VII (236 ml/kg; 175 ml/kg, measured by activated factor VII) as compared to plasma-derived factor VII (206 ml/kg; 64 ml/kg, measured by factor FVII activity), suggesting a plasma- and extracellular fluid distribution for recombinant activated factor VII. Recombinant activated factor VII showed significantly shorter half-lifes than plasma-derived factor VII. Volumes of distribution were significantly higher for treatment with recombinant activated factor VII. The longer half-life for plasma-derived factor VII, compared to recombinant activated factor VII, and the increased volume of distribution for recombinant activated factor VII, compared to plasma-derived factor VII may further elucidate the beneficial effect of prophylactic treatment of both products. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Molecular characterization of cathepsin B from Clonorchis sinensis excretory/secretory products and assessment of its potential for serodiagnosis of clonorchiasis

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    Zhou Chenhui

    2011-07-01

    Full Text Available Abstract Background Cathepsin cysteine proteases play multiple roles in the life cycle of parasites such as food uptake, immune invasion and pathogenesis, making them valuable targets for diagnostic assays, vaccines and drugs. The purpose of this study was to identify a cathepsin B of Clonorchis sinensis (CsCB and to investigate its diagnostic value for human helminthiases. Results The predicted amino acid sequence of the cathepsin B of C. sinensis shared 63%, 52%, 50% identity with that of Schistosoma japonicum, Homo sapiens and Fasciola hepatica, respectively. Sequence encoding proenzyme of CsCB was overexpressed in Escherichia coli. Reverse transcription PCR experiments revealed that CsCB transcribed in both adult worm and metacercaria of C. sinensis. CsCB was identified as a C. sinensis excretory/secretory product by immunoblot assay, which was consistent with immunohistochemical localization showing that CsCB was especially expressed in the intestine of C. sinensis adults. Both ELISA and western blotting analysis showed recombinant CsCB could react with human sera from clonorchiasis and other helminthiases. Conclusions Our findings revealed that secreted CsCB may play an important role in the biology of C. sinensis and could be a diagnostic candidate for helminthiases.

  2. A new impedance based approach to test the activity of recombinant protein--Semaphorins as a test case.

    Science.gov (United States)

    Birger, Anastasya; Besser, Elazar; Reubinoff, Benjamin; Behar, Oded

    2015-10-01

    The biological activity of a recombinant protein is routinely measured using a bioassay such as an enzyme assay. However, many proteins have no enzymatic activity and in many cases it is difficult to devise a simple and reliable approach to test their activity. Semaphorins, Ephrins, Slits, Netrins or amylin-assisted proteins have numerous activities affecting many systems and cell types in the human body. Most of them are also able to induce rapid cytoskeleton changes at least in some cell types. We assumed therefore, that such proteins might be tested based on their ability to modulate the cytoskeleton. Here we tested a number of semaphorins in an impedance based label-free platform that allows for dynamic monitoring of subtle morphological and adhesive changes. This system has proved to be a very fast, sensitive and effective way to monitor and determine the activity of such proteins. Furthermore we showed that it is possible to customize a cell-protein system by transfecting the cells with specific receptors and test the cell response following the addition of the recombinant ligand protein. Since other protein families such as Ephrins and Netrins can also influence the cytoskeleton of some cells, this approach may be applicable to a large number of proteins. Copyright © 2015 Elsevier GmbH. All rights reserved.

  3. Superoxide dismutase recombinant Lactobacillus fermentum ameliorates intestinal oxidative stress through inhibiting NF-κB activation in a trinitrobenzene sulphonic acid-induced colitis mouse model.

    Science.gov (United States)

    Hou, C L; Zhang, J; Liu, X T; Liu, H; Zeng, X F; Qiao, S Y

    2014-06-01

    Superoxide dismutase (SOD) can prevent and cure inflammatory bowel diseases by decreasing the amount of reactive oxygen species. Unfortunately, short half-life of SOD in the gastrointestinal tract limited its application in the intestinal tract. This study aimed to investigate the treatment effects of recombinant SOD Lactobacillus fermentum in a colitis mouse model. In this study, we expressed the sodA gene in Lact. fermentum I5007 to obtain the SOD recombinant strain. Then, we determined the therapeutic effects of this SOD recombinant strain in a trinitrobenzene sulphonic acid (TNBS)-induced colitis mouse model. We found that SOD activity in the recombinant Lact. fermentum was increased by almost eightfold compared with that in the wild type. Additionally, both the wild type and the recombinant Lact. fermentum increased the numbers of lactobacilli in the colon of mice (P < 0·05). Colitis mice treated with recombinant Lact. fermentum showed a higher survival rate and lower disease activity index (P < 0·05). Recombinant Lact. fermentum significantly decreased colonic mucosa histological scoring for infiltration of inflammatory cells, lipid peroxidation, the expression of pro-inflammatory cytokines and myeloperoxidase (P < 0·05) and inhibited NF-κB activity in colitis mice (P < 0·05). SOD recombinant Lact. fermentum significantly reduced oxidative stress and inflammation through inhibiting NF-κB activation in the TNBS-induced colitis model. This study provides insights into the anti-inflammatory effects of SOD recombinant Lact. fermentum, indicating the potential therapeutic effects in preventing and curing intestinal bowel diseases. © 2014 The Society for Applied Microbiology.

  4. Cathepsin O is involved in the innate immune response and metamorphosis of Antheraea pernyi.

    Science.gov (United States)

    Sun, Yu-Xuan; Zhu, Bao-Jian; Tang, Lin; Sun, Yu; Chen, Chen; Nadeem Abbas, Muhammad; Wang, Lei; Qian, Cen; Wei, Guo-Qing; Liu, Chao-Liang

    2017-11-01

    Cathepsins are key members of mammalian papain-like cysteine proteases that play an important role in the immune response. In this study, a fragment of cDNA encoding cathepsin O proteinase (ApCathepsin O) was cloned from Antheraea pernyi. It contains an open reading frame of 1170bp and encodes a protein with 390 amino acid residues, including a conserved I29 inhibitor domain and a peptidase C1A (clan CA of cysteine proteases, papain family C1 subfamily) domain. Comparison with other previously reported cathepsin O proteins showed identity ranging from 45% to 79%. Quantitative real-time PCR (qRT-PCR) and Western blot analysis revealed that ApCathepsin O was highly expressed in the fat body; furthermore, the high expression during the pupal stage indicated that it might be involved during metamorphosis. After exposure to four different heat-killed pathogens (Escherichia coli, Beauveria bassiana, Micrococcus luteus, and A. pernyi nucleopolyhedrovirus), the expression levels of ApCathepsin O mRNA significantly increased and showed variable expression patterns. This indicates that ApCathepsin O is potentially involved in the innate immune system of A. pernyi. Interestingly, ApCathepsin O expression was upregulated after 20-hydroxyecdysone (20E) injection, which suggested that it might be regulated by 20E. In conclusion, ApCathepsin O is a protease that may play an important role in the innate immune response and metamorphosis of A. pernyi. Copyright © 2017. Published by Elsevier Inc.

  5. Vaccination against Fasciola hepatica using cathepsin L3 and B3 proteases delivered alone or in combination.

    Science.gov (United States)

    Wesołowska, Agnieszka; Basałaj, Katarzyna; Norbury, Luke J; Sielicka, Alicja; Wędrychowicz, Halina; Zawistowska-Deniziak, Anna

    2018-01-30

    No licensed vaccine is currently available for prevention of Fasciola hepatica infections. However, considering the alarming increase in drug resistance, there is an urgent need for a safe and fully effective vaccine against fasciolosis. Here, we tested if cathepsins L (FhCL3-1, FhCL3-2) and B (FhCB3) secreted by juvenile liver flukes are viable vaccine targets when delivered alone or in combination in a rat model. Since control over the early immune response is crucial for parasite's establishment in its host, it was hypothesised that targeting fluke juvenile stages may prove beneficial. Moreover, it was assumed that selected antigens will act in a cumulative manner to interfere with liver fluke migration and thereby will reduce F. hepatica infection. Recombinant FhCL3-1 and FhCL3-2 delivered alone reduced liver fluke burdens by 47 % and 63 %, respectively. A trivalent vaccine containing rFhCL3-1/CL3-2/CB3 did not increase the protective vaccine efficacy compared to the rFhCL3-2 vaccinated group (53 %), although, reductions in liver fluke wet weight (statistically significant) and liver damage score were most pronounced. Further, the highest IgG1 and IgG2a levels were seen in rFhCL3-2 vaccinated rats, the group for which the highest reduction in worm burden was demonstrated. Moreover, IgG1 and IgG2a levels in vaccinated rats were significantly elevated compared to those reported for control groups up to 4 week post-infection. While the mechanism of protection remains unknown, it appears that it depends on vaccine-induced antibodies directed against cathepsins. The obtained results imply that F. hepatica juvenile-specific cathepsins are promising vaccine candidates that induce responses that successfully target early migratory liver fluke stages. Now, the challenge is to evaluate these juvenile-specific cathepsins for use in livestock. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. The Potential Role of the Proteases Cathepsin D and Cathepsin L in the Progression and Metastasis of Epithelial Ovarian Cancer

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    Md Zahidul Islam Pranjol

    2015-11-01

    Full Text Available Epithelial ovarian cancer (EOC is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

  7. The Potential Role of the Proteases Cathepsin D and Cathepsin L in the Progression and Metastasis of Epithelial Ovarian Cancer.

    Science.gov (United States)

    Pranjol, Md Zahidul Islam; Gutowski, Nicholas; Hannemann, Michael; Whatmore, Jacqueline

    2015-11-20

    Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancies and has a poor prognosis due to relatively unspecific early symptoms, and thus often advanced stage, metastasized cancer at presentation. Metastasis of EOC occurs primarily through the transcoelomic route whereby exfoliated tumor cells disseminate within the abdominal cavity, particularly to the omentum. Primary and metastatic tumor growth requires a pool of proangiogenic factors in the microenvironment which propagate new vasculature in the growing cancer. Recent evidence suggests that proangiogenic factors other than the widely known, potent angiogenic factor vascular endothelial growth factor may mediate growth and metastasis of ovarian cancer. In this review we examine the role of some of these alternative factors, specifically cathepsin D and cathepsin L.

  8. 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.

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

    Science.gov (United States)

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

    2016-01-01

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

  10. The 3'-to-5' exonuclease activity of vaccinia virus DNA polymerase is essential and plays a role in promoting virus genetic recombination.

    Science.gov (United States)

    Gammon, Don B; Evans, David H

    2009-05-01

    Poxviruses are subjected to extraordinarily high levels of genetic recombination during infection, although the enzymes catalyzing these reactions have never been identified. However, it is clear that virus-encoded DNA polymerases play some unknown yet critical role in virus recombination. Using a novel, antiviral-drug-based strategy to dissect recombination and replication reactions, we now show that the 3'-to-5' proofreading exonuclease activity of the viral DNA polymerase plays a key role in promoting recombination reactions. Linear DNA substrates were prepared containing the dCMP analog cidofovir (CDV) incorporated into the 3' ends of the molecules. The drug blocked the formation of concatemeric recombinant molecules in vitro in a process that was catalyzed by the proofreading activity of vaccinia virus DNA polymerase. Recombinant formation was also blocked when CDV-containing recombination substrates were transfected into cells infected with wild-type vaccinia virus. These inhibitory effects could be overcome if CDV-containing substrates were transfected into cells infected with CDV-resistant (CDV(r)) viruses, but only when resistance was linked to an A314T substitution mutation mapping within the 3'-to-5' exonuclease domain of the viral polymerase. Viruses encoding a CDV(r) mutation in the polymerase domain still exhibited a CDV-induced recombination deficiency. The A314T substitution also enhanced the enzyme's capacity to excise CDV molecules from the 3' ends of duplex DNA and to recombine these DNAs in vitro, as judged from experiments using purified mutant DNA polymerase. The 3'-to-5' exonuclease activity appears to be an essential virus function, and our results suggest that this might be because poxviruses use it to promote genetic exchange.

  11. Whole-genome analysis of genetic recombination of hepatitis delta virus: molecular domain in delta antigen determining trans-activating efficiency.

    Science.gov (United States)

    Chao, Mei; Lin, Chia-Chi; Lin, Feng-Ming; Li, Hsin-Pai; Iang, Shan-Bei

    2015-12-01

    Hepatitis delta virus (HDV) is the only animal RNA virus that has an unbranched rod-like genome with ribozyme activity and is replicated by host RNA polymerase. HDV RNA recombination was previously demonstrated in patients and in cultured cells by analysis of a region corresponding to the C terminus of the delta antigen (HDAg), the only viral-encoded protein. Here, a whole-genome recombination map of HDV was constructed using an experimental system in which two HDV-1 sequences were co-transfected into cultured cells and the recombinants were analysed by sequencing of cloned reverse transcription-PCR products. Fifty homologous recombinants with 60 crossovers mapping to 22 junctions were identified from 200 analysed clones. Small HDAg chimeras harbouring a junction newly detected in the recombination map were then constructed. The results further indicated that the genome-replication level of HDV was sensitive to the sixth amino acid within the N-terminal 22 aa of HDAg. Therefore, the recombination map established in this study provided a tool for not only understanding HDV RNA recombination, but also elucidating the related mechanisms, such as molecular elements responsible for the trans-activation levels of the small HDAg.

  12. Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing.

    Science.gov (United States)

    Khaki, Mohsen; Salmanian, Ali Hatef; Mosayebi, Ghasem; Baazm, Maryam; Babaei, Saeed; Molaee, Neda; Abtahi, Hamid

    2017-07-01

    Vascular endothelial growth factor (VEGF) is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs) differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli ( E. coli ) system and then biological activity of this protein was evaluated in animal wound healing. E. coli BL21 (DE3) competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG). The recombinant protein was purified by affinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w) was used for external wound (25×15mm thickness) healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. The recombinant protein with molecular weight of 45 kilodaltons (kDa) and concentration of 0.8 mg/ml was produced. Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Recombinant VEGF-A produced by pET32a in E. coli , possesses acceptable structure and has wound healing capability.

  13. Heterologous expression of a truncated form of human recombinant vascular endothelial growth factor-A and its biological activity in wound healing

    Directory of Open Access Journals (Sweden)

    Mohsen Khaki

    2017-07-01

    Full Text Available Objective(s: Vascular endothelial growth factor (VEGF is one of the most effective proteins in angiogenesis, mesenchymal stem cells (MSCs differentiation and wound healing. These abilities are therapeutic potential of VEGF in diabetic retinopathy, nephropathy and other tissue damage circumstances. In this study, recombinant VEGF was produced in Escherichia coli (E. coli system and then biological activity of this protein was evaluated in animal wound healing. Materials and Methods: E. coli BL21 (DE3 competent cells were transformed with pET32a-VEGF clone and induced by isopropyl-β-D-thio-galactoside (IPTG. The recombinant protein was purified byaffinity chromatography. Recombinant VEGF-A-based ointment (VEGF/Vaseline 0.8 mg/100 w/w was used for external wound (25×15mm thickness healing in animal model. In vivo activity of ointment was evaluated by clinical evidences and cytological microscopic assessment. Results: The recombinant protein with molecular weight of 45 kilodaltons (kDa and concentration of 0.8 mg/ml was produced.Immunoblotting data showed that the antigenic region of VEGF can be expressed in E. coli and the recombinant protein has similar epitopes with close antigenic properties to the natural form. Macroscopic findings and microscopic data showed that the recombinant VEGF-A ointment was effective on excisional wound healing. Conclusion: Recombinant VEGF-A produced by pET32a in E. coli, possesses acceptable structure and has wound healing capability.

  14. Increased Plasma Cathepsin S at the Time of Percutaneous Transluminal Angioplasty is Associated with 6-Months’ Restenosis of the Femoropopliteal Artery

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    Mijovski Mojca Bozic

    2018-01-01

    Full Text Available Background: We tested the hypothesis that increased levels of cathepsin S and decreased levels of cystatin C in plasma at the time of percutaneous transluminal angioplasty (PTA are associated with the occurrence of 6-months’ restenosis of the femoropopliteal artery (FPA. Methods: 20 patients with restenosis and 24 matched patients with patent FPA after a 6-months follow-up were in - cluded in this study. They all exhibited disabling claudication or critical limb ischemia and had undergone technically successful PTA. They were all receiving statins and ACE in hi - bitors (or angiotensin II receptor antagonist before the PTA and the therapy did not change throughout the observational period. Plasma concentrations of C-reactive protein were < 10 mg/L and of creatinine within the reference range at the time of the PTA. Plasma concentration and activity of cathepsin S, together with its potent inhibitor cystatin C, were measured the day before and the day after the PTA. Results: The increased plasma concentration and activity of cathepsin S at the time of PTA was associated with the occurrence of 6-months’ restenosis of FPA, independently of established risk factors (lesion complexity, infrapopliteal run-off vessels, type of PTA, age, gender, smoking, diabetes, lipids and of cystatin C. Plasma cystatin C concentration was not associated with restenosis and did not correlate with cathepsin S activity and concentration in the plasma. Conclusion: Increased level of plasma cathepsin S at the time of PTA is associated with 6-months’ restenosis of PTA, independently of established risk factors.

  15. Outcome of stroke patients receiving different doses of recombinant tissue plasminogen activator.

    Science.gov (United States)

    Ong, Cheung-Ter; Wong, Yi-Sin; Wu, Chi-Shun; Su, Yu-Hsiang

    2017-01-01

    Intravenous recombinant tissue plasminogen activator (tPA) at a dose of 0.9 mg/kg body weight is associated with a high hemorrhagic transformation (HT) rate. Low-dose tPA (0.6 mg/kg) may have a lower hemorrhage rate but the mortality and disability rates at 90 days cannot be confirmed as non-inferior to standard-dose tPA. Whether the doses 0.7 and 0.8 mg/kg have better efficacy and safety needs further investigation. Therefore, this study is to compare the efficacy and safety of each dose of tPA (0.6, 0.7, 0.8, and 0.9 mg/kg body weight) and to investigate the factors affecting early neurological improvement (ENI) and early neurological deterioration (END). For this observational study, data were obtained from 274 patients who received tPA thrombolytic therapy in Chia-Yi Christian Hospital stroke unit. The tPA dose was given at the discretion of each physician. The definition of ENI was a >8 point improvement (compared with baseline) at 24 h following thrombolytic therapy or an improvement in the National Institutes of Health Stroke Score (NIHSS) to 0 or 1 toward the end of tPA infusion. The definition of END was a >4 point increase in NIHSS (compared with baseline) within 24 h of tPA infusion. The primary objective was to investigate whether 0.7 and 0.8 mg/kg of tPA have higher ENI rate, lower END rate, and better outcome at 6 months. Poor outcome was defined as having a modified Rankin Scale of 3 to 6 (range, 0 [no symptoms] to 6 [death]). The secondary objective was to investigate whether low-dose tPA has a lower risk of intracerebral HT than that with standard-dose tPA. We also investigated the factors affecting ENI, END, HT, and 6-month outcome. A total of 274 patients were included during the study period, of whom 260 were followed up for >6 months. There was a trend for the HT rate to increase as the dose increased ( P =0.02). The symptomatic HT rate was not significantly different among the low-dose and standard-dose groups. The ENI and END ( P =0.52) were

  16. Continuous infusion of recombinant activated factor VII for bleeding control after lobectomy in a patient with inherited factor VII deficiency.

    Science.gov (United States)

    Miyata, Naoko; Isaka, Mitsuhiro; Kojima, Hideaki; Maniwa, Tomohiro; Takahashi, Shoji; Takamiya, Osamu; Ohde, Yasuhisa

    2016-03-01

    Inherited factor VII (FVII) deficiency is a rare recessive inherited coagulation disorder with limited available information, especially in patients undergoing major thoracic surgery. In addition, an optimal management strategy for the disease has not been defined. We herein report a case involving a 61-year-old man with asymptomatic FVII deficiency who underwent a right middle and lower lobectomy to treat lung cancer. To the best of our knowledge, the present report is the first to describe the use of recombinant activated FVII continuous infusion for bleeding control after a major thoracic surgery in a patient with inherited FVII deficiency.

  17. Effect of some factors used to the chicken meat preservation and processing on the protease activity

    International Nuclear Information System (INIS)

    Przysiezna, E.; Skrabka-Blotnicka, T.

    1996-01-01

    The obtained results indicated that the cathepsin activity was higher by about 60% in the extract from thigh than from breast muscles. Freezing and defrosting (not stored) of chicken meat did not influence the breast muscle cathepsin activity while they caused a decrease of activity of about 20% in the case of thigh muscles. The increase in cathepsin activity was noticed in both kinds of muscles during storage at -20 °C up to 4 months (45.6% and 19.4% for thigh and breast muscles respectively). The activity of cathepsin in extract from 5 months stored meat reached 80% in case of breast muscles and 83% in case of thigh muscles in relation to control sample respectively. The cathepsin activity significantly increased during heating of breast muscles up to 60 °C, but in case of thigh muscles it was slightly higher than at 50°C. The heating of cured chicken breast muscles up to 60°C caused a non significant growth in cathepsin activity opposite to raw muscles. The cathepsin activity from all cured samples heated up to 70°C were several times lower in relation to control samples. The cathepsin activity of both thigh and breast muscles were resistant to gamma radiation. The investigated factors caused changes in the activity of cathepsin but none of them caused its total inactivation. The changes of cathepsin activity depended on the kind of muscles and the kind and the value of acting factors

  18. A Novel Cold-Active Lipase from Candida albicans: Cloning, Expression and Characterization of the Recombinant Enzyme

    Directory of Open Access Journals (Sweden)

    Dong-Ming Lan

    2011-06-01

    Full Text Available A novel lipase gene lip5 from the yeast Candida albicans was cloned and sequenced. Alignment of amino acid sequences revealed that 86–34% identity exists with lipases from other Candida species. The lipase and its mutants were expressed in the yeast Pichia pastoris, where alternative codon usage caused the mistranslation of 154-Ser and 293-Ser as leucine. 154-Ser to leucine resulted in loss of expression of Lip5, and 293-Ser to leucine caused a marked reduction in the lipase activity. Lip5-DM, which has double mutations that revert 154 and 293 to serine residues, showed good lipase activity, and was overexpressed and purified by (NH42SO4 precipitation and ion-exchange chromatography. The pure Lip5-DM was stable at low temperatures ranging from 15–35 °C and pH 5–9, with the optimal conditions being 15–25 °C and pH 5–6. The activation energy of recombinant lipase was 8.5 Kcal/mol between 5 and 25 °C, suggesting that Lip5-DM was a cold–active lipase. Its activity was found to increase in the presence of Zn2+, but it was strongly inhibited by Fe2+, Fe3+, Hg2+ and some surfactants. In addition, the Lip5-DM could not tolerate water-miscible organic solvents. Lip5-DM exhibited a preference for the short- and medium-chain length p-nitrophenyl (C4 and C8 acyl group esters rather than the long chain length p-nitrophenyl esters (C12, C16 and C18 acyl group with highest activity observed with the C8 derivatives. The recombinant enzyme displayed activity toward triacylglycerols, such as olive oil and safflower oil.

  19. Cathepsin B Improves ß-Amyloidosis and Learning and Memory in Models of Alzheimer's Disease.

    Science.gov (United States)

    Embury, Christine M; Dyavarshetty, Bhagyalaxmi; Lu, Yaman; Wiederin, Jayme L; Ciborowski, Pawel; Gendelman, Howard E; Kiyota, Tomomi

    2017-06-01

    Amyloid-ß (Aß) precursor protein (APP) metabolism engages neuronal endolysosomal pathways for Aß processing and secretion. In Alzheimer's disease (AD), dysregulation of APP leads to excess Aß and neuronal dysfunction; suggesting that neuronal APP/Aß trafficking can be targeted for therapeutic gain. Cathepsin B (CatB) is a lysosomal cysteine protease that can lower Aß levels. However, whether CatB-modulation of Aß improves learning and memory function deficits in AD is not known. To this end, progenitor neurons were infected with recombinant adenovirus expressing CatB and recovered cell lysates subjected to proteomic analyses. The results demonstrated Lamp1 deregulation and linkages between CatB and the neuronal phagosome network. Hippocampal injections of adeno-associated virus expressing CatB reduced Aß levels, increased Lamp1 and improved learning and memory. The findings were associated with the emergence of c-fos + cells. The results support the idea that CatB can speed Aß metabolism through lysosomal pathways and as such reduce AD-associated memory deficits.

  20. The glycoprotein Ib-IX-V complex contributes to tissue factor-independent thrombin generation by recombinant factor VIIa on the activated platelet surface

    NARCIS (Netherlands)

    Weeterings, Cees; de Groot, Philip G.; Adelmeijer, Jelle; Lisman, Ton

    2008-01-01

    Several lines of evidence suggest that recombinant factor VIIa (rFVIIa) is able to activate factor X on an activated platelet, in a tissue factor-independent manner. We hypothesized that, besides the anionic surface, a receptor on the activated platelet surface is involved in this process. Here, we

  1. PROTECTIVE ACTIVITY STUDY OF A CANDIDATE VACCINE AGAINST ROTAVIRUS INFECTION BASED ON RECOMBINANT PROTEIN FliCVP6VP8

    Directory of Open Access Journals (Sweden)

    I. V. Dukhovlinov

    2016-01-01

    Full Text Available Rotavirus infection is among leading causes of severe diarrhea which often leads to severe dehydration, especially, in children under 5 years old. In Russia, the incidence of rotavirus infection is constantly increased, due to higher rates of actual rotavirus infection cases and improved diagnostics of the disease. Immunity to rotavirus is unstable, thus causing repeated infections intra vitam. Anti-infectious resistance in reconvalescents is explained by induction of specific IgM, IgG, and, notably, IgA antibodies. Due to absence of market drugs with direct action against rotavirus, a rational vaccination is considered the most effective way to control the disease. Currently available vaccines for prevention of rotavirus infection are based on live attenuated rotavirus strains, human and/or animal origin, which replicate in human gut. Their implementation may result into different complications. Meanwhile, usage of vaccines based on recombinant proteins is aimed to avoid risks associated with introduction of a complete virus into humans. In this paper, we studied protective activity of candidate vaccines against rotavirus.In this work we studied protective activity of a candidate vaccine against rotavirus infection based on recombinant FliCVP6VP8 protein which includes VP6 and VP8, as well as components of Salmonella typhimurium flagellin (FliC as an adjuvant. Different components are joined by flexible bridges. Efficiency of the candidate vaccine was studied in animal model using Balb/c mice. We have shown high level of protection which occurs when the candidate vaccine is administered twice intramuscularly. Complete protection of animals against mouse rotavirus EDC after intramuscular immunization with a candidate vaccine was associated with arising rotavirus-specific IgA and IgG antibodies in serum and intestine of immunized animals. The efficacy of candidate vaccine based on recombinant protein FliCVP6VP8 against rotavirus infection was

  2. Fibrinogen and fibrin are novel substrates for Fasciola hepatica cathepsin L peptidases

    NARCIS (Netherlands)

    Mebius, Mirjam M.; Op Heij, Jody M J; Tielens, Aloysius G.M.; de Groot, Philip G; Urbanus, Rolf T; van Hellemond, Jaap J.

    2018-01-01

    Cathepsin peptidases form a major component of the secreted proteins of the blood-feeding trematodes Fasciola hepatica and Schistosoma mansoni. These peptidases fulfill many functions, from facilitating infection to feeding and immune evasion. In this study, we examined the Fasciola cathepsin L

  3. Identification of Chalcones as Fasciola hepatica Cathepsin L Inhibitors Using a Comprehensive Experimental and Computational Approach.

    Directory of Open Access Journals (Sweden)

    Florencia Ferraro

    2016-07-01

    Full Text Available Increased reports of human infections have led fasciolosis, a widespread disease of cattle and sheep caused by the liver flukes Fasciola hepatica and Fasciola gigantica, to be considered an emerging zoonotic disease. Chemotherapy is the main control measure available, and triclabendazole is the preferred drug since is effective against both juvenile and mature parasites. However, resistance to triclabendazole has been reported in several countries urging the search of new chemical entities and target molecules to control fluke infections.We searched a library of forty flavonoid derivatives for inhibitors of key stage specific Fasciola hepatica cysteine proteases (FhCL3 and FhCL1. Chalcones substituted with phenyl and naphtyl groups emerged as good cathepsin L inhibitors, interacting more frequently with two putative binding sites within the active site cleft of the enzymes. One of the compounds, C34, tightly bounds to juvenile specific FhCL3 with an IC50 of 5.6 μM. We demonstrated that C34 is a slow-reversible inhibitor that interacts with the Cys-His catalytic dyad and key S2 and S3 pocket residues, determinants of the substrate specificity of this family of cysteine proteases. Interestingly, C34 induces a reduction in NEJ ability to migrate through the gut wall and a loss of motility phenotype that leads to NEJ death within a week in vitro, while it is not cytotoxic to bovine cells.Up to date there are no reports of in vitro screening for non-peptidic inhibitors of Fasciola hepatica cathepsins, while in general these are considered as the best strategy for in vivo inhibition. We have identified chalcones as novel inhibitors of the two main Cathepsins secreted by juvenile and adult liver flukes. Interestingly, one compound (C34 is highly active towards the juvenile enzyme reducing larval ability to penetrate the gut wall and decreasing NEJ´s viability in vitro. These findings open new avenues for the development of novel agents to control

  4. Assessment of the adjuvant activity of mesoporous silica nanoparticles in recombinant Mycoplasma hyopneumoniae antigen vaccines

    Directory of Open Access Journals (Sweden)

    Veridiana Gomes Virginio

    2017-01-01

    Full Text Available The adjuvant potential of two mesoporous silica nanoparticles (MSNs, SBa-15 and SBa-16, was assessed in combination with a recombinant HSP70 surface polypeptide domain from Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia (PEP. The recombinant antigen (HSP70212-600, previously shown as immunogenic in formulation with classic adjuvants, was used to immunize BALB/c mice in combination with SBa-15 or SBa-16 MSNs, and the effects obtained with these formulations were compared to those obtained with alum, the adjuvant traditionally used in anti-PEP bacterins. The HSP70212-600 + SBa-15 vaccine elicited a strong humoral immune response, with high serum total IgG levels, comparable to those obtained using HSP70212-600 + alum. The HSP70212-600 + SBa-16 vaccine elicited a moderate humoral immune response, with lower levels of total IgG. The cellular immune response was assessed by the detection of IFN-γ, IL-4 and IL-10 in splenocyte culture supernatants. The HSP70212-600 + SBa-15 vaccine increased IFN-γ, IL-4 and IL-10 levels, while no stimulation was detected with the HSP70212-600 + SBa-16 vaccine. The HSP70212-600 + SBa-15 vaccine induced a mixed Th1/Th2-type response, with an additional IL-10 mediated anti-inflammatory effect, both of relevance for an anti-PEP vaccine. Alum adjuvant controls stimulated an unspecific cellular immune response, with similar levels of cytokines detected in mice immunized either with HSP70212-600 + alum or with the adjuvant alone. The better humoral and cellular immune responses elicited in mice indicated that SBa-15 has adjuvant potential, and can be considered as an alternative to the use of alum in veterinary vaccines. The use of SBa-15 with HSP70212-600 is also promising as a potential anti-PEP subunit vaccine formulation.

  5. Serum cathepsin H as a potential prognostic marker in patients with colorectal cancer

    DEFF Research Database (Denmark)

    Schweiger, A; Christensen, Ib Jarle; Nielsen, Hans Jørgen

    2005-01-01

    Cathepsin H is a lysosomal cysteine protease that may participate in tumor progression. In order to evaluate its potential as a prognostic marker, its protein levels were measured by ELISA in preoperative sera from 324 patients with colorectal cancer. The level of cathepsin H was significantly...... increased in patient sera, the median level was 8.4 ng/mL versus 2.1 ng/mL in 90 healthy blood donors (p CEA). In survival analysis...... a significant difference was found between the group of patients with low cathepsin H (first tertile) who had a poor prognosis and the remaining patients (p = 0.03). The risk of patients was further stratified when cathepsin H levels were combined with CEA. Patients with high CEA and low cathepsin H had...

  6. Synthesis and biochemical evaluation of benzoylbenzophenone thiosemicarbazone analogues as potent and selective inhibitors of cathepsin L

    DEFF Research Database (Denmark)

    Parker, Erica N; Song, Jiangli; Kishore Kumar, G D

    2015-01-01

    selective in their inhibition of cathepsin L compared to cathepsin B. Thiosemicarbazone analogue 32 inhibited invasion through Matrigel of MDA-MB-231 breast cancer cells by 70% at 10μM. Thiosemicarbazone analogue 8 significantly inhibited the invasive potential of PC-3ML prostate cancer cells by 92% at 5μ......Upregulation of cathepsin L in a variety of tumors and its ability to promote cancer cell invasion and migration through degradation of the extracellular matrix suggest that cathepsin L is a promising biological target for the development of anti-metastatic agents. Based on encouraging results from......) was well-tolerated in a CDF1 mouse model bearing an implanted C3H mammary carcinoma, and showed efficacy in tumor growth delay. Low cytotoxicity, inhibition of cell invasion, and in vivo tolerability are desirable characteristics for anti-metastatic agents functioning through an inhibition of cathepsin L...

  7. Juvenile-specific cathepsin proteases in Fasciola spp.: their characteristics and vaccine efficacies.

    Science.gov (United States)

    Meemon, Krai; Sobhon, Prasert

    2015-08-01

    Fasciolosis, caused by Fasciola hepatica and Fasciola gigantica, is one of the most neglected tropical zoonotic diseases. One sustainable control strategy against these infections is the employment of vaccines that target proteins essential for parasites' invasion and nutrition acquiring processes. Cathepsin proteases are the most abundantly expressed proteins in Fasciola spp. that have been tested successfully as vaccines against fasciolosis in experimental as well as large animals because of their important roles in digestion of nutrients, invasion, and migration. Specifically, juvenile-specific cathepsin proteases are the more effective vaccines because they could block the invasion and migration of juvenile parasites whose immune evasion mechanism has not yet been fully developed. Moreover, because of high sequence similarity and identity of cathepsins from juveniles with those of adults, the vaccines can attack both the juvenile and adult stages. In this article, the characteristics and vaccine potentials of juvenile-specific cathepsins, i.e., cathepsins L and B, of Fasciola spp. were reviewed.

  8. Prediction of Aggressive Human Prostate Cancer by Cathepsin B

    Science.gov (United States)

    2008-03-01

    Cancer Res 2004;10(12 Pt 1):4118-4124. 28. Munoz E, Gomez F, Paz JI, Casado I, Silva JM, Corcuera MT, Alonso MJ. Ki-67 immunolabeling in pre...detected prostate cancer. J Pathol 2002;197(2):148-154. 34. Claudio PP, Zamparelli A, Garcia FU, Claudio L, Ammirati G, Farina A, Bovicelli A, Russo G...JA. Distinct roles for cysteine cathepsin genes in multistage tumorigenesis. Genes Dev 2006;20(5):543-556. 47. Fernandez PL, Farre X, Nadal A

  9. Genetic Recombination

    Science.gov (United States)

    Whitehouse, H. L. K.

    1973-01-01

    Discusses the mechanisms of genetic recombination with particular emphasis on the study of the fungus Sordaria brevicollis. The study of recombination is facilitated by the use of mutants of this fungus in which the color of the ascospores is affected. (JR)

  10. Nanocrystalline NdFeB magnet prepared by mechanically activated disproportionation and desorption-recombination in-situ sintering

    International Nuclear Information System (INIS)

    Xiaoya, Liu; Yuping, Li; Lianxi, Hu

    2013-01-01

    The process of mechanically activated disproportionation and desorption-recombination in-situ sintering was proposed to synthesize highly densified nanocrystalline NdFeB magnet, and its validity was demonstrated by experimental investigation with the use of a Nd 16 Fe 76 B 8 (atomic ratio) alloy. Firstly, the as-cast alloy was disproportionated by mechanical milling in hydrogen, with the starting micron-sized Nd 2 Fe 14 B phase decomposed into an intimate mixture of nano-structured NdH 2.7 , Fe 2 B and α-Fe phases. The as-disproportionated alloy powders were compacted by cold pressing and then subjected to desorption-recombination in-situ sintering. The microstructure of both the as-disproportionated and the subsequently sintered samples was characterized by X-ray diffraction and electron transmission microscopy, respectively. The magnetic properties of the sintered samples were measured by using vibrating sample magnetometer. The results showed that, by vacuum sintering, not only was the powder compact consolidated, but also the as-disproportionated microstucture transformed into nanocrystalline Nd 2 Fe 14 B phase via the well-known desorption-recombination reaction, thus giving rise to nanocrystalline NdFeB magnet. In the present study, the optimal sintering parameters were found to be 780 °C×30 min. In this case, the coercivity, the remanence, and maximum energy product of the magnet sample achieved 0.8 T, 635.3 kA/m, and 106.3 kJ/m 3 , respectively. - Highlights: ► Nano-structured disproportionated NdFeB alloy powders by mechanical milling in hydrogen. ► Highly densified green magnet compact by cold pressing of as-disproportionated NdFeB alloy powders. ► Nanocrystalline NdFeB magnets by desorption-recombination in-situ sintering under vacuum. ► Magnetic properties significantly improved by relative density enhancement and nanocrystallization of Nd 2 Fe 14 B phase. ► The effects of sintering parameters on magnetic properties and the underlying

  11. Nanocrystalline NdFeB magnet prepared by mechanically activated disproportionation and desorption-recombination in-situ sintering

    Energy Technology Data Exchange (ETDEWEB)

    Xiaoya, Liu; Yuping, Li [School of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001 (China); Lianxi, Hu, E-mail: hulx@hit.edu.cn [School of Materials Science and Engineering, Harbin Institute of Technology, Harbin 150001 (China)

    2013-03-15

    The process of mechanically activated disproportionation and desorption-recombination in-situ sintering was proposed to synthesize highly densified nanocrystalline NdFeB magnet, and its validity was demonstrated by experimental investigation with the use of a Nd{sub 16}Fe{sub 76}B{sub 8} (atomic ratio) alloy. Firstly, the as-cast alloy was disproportionated by mechanical milling in hydrogen, with the starting micron-sized Nd{sub 2}Fe{sub 14}B phase decomposed into an intimate mixture of nano-structured NdH{sub 2.7}, Fe{sub 2}B and {alpha}-Fe phases. The as-disproportionated alloy powders were compacted by cold pressing and then subjected to desorption-recombination in-situ sintering. The microstructure of both the as-disproportionated and the subsequently sintered samples was characterized by X-ray diffraction and electron transmission microscopy, respectively. The magnetic properties of the sintered samples were measured by using vibrating sample magnetometer. The results showed that, by vacuum sintering, not only was the powder compact consolidated, but also the as-disproportionated microstucture transformed into nanocrystalline Nd{sub 2}Fe{sub 14}B phase via the well-known desorption-recombination reaction, thus giving rise to nanocrystalline NdFeB magnet. In the present study, the optimal sintering parameters were found to be 780 Degree-Sign C Multiplication-Sign 30 min. In this case, the coercivity, the remanence, and maximum energy product of the magnet sample achieved 0.8 T, 635.3 kA/m, and 106.3 kJ/m{sup 3}, respectively. - Highlights: Black-Right-Pointing-Pointer Nano-structured disproportionated NdFeB alloy powders by mechanical milling in hydrogen. Black-Right-Pointing-Pointer Highly densified green magnet compact by cold pressing of as-disproportionated NdFeB alloy powders. Black-Right-Pointing-Pointer Nanocrystalline NdFeB magnets by desorption-recombination in-situ sintering under vacuum. Black-Right-Pointing-Pointer Magnetic properties significantly

  12. Purification and characterization of cathepsin D from herring muscle ( Clupea harengus )

    DEFF Research Database (Denmark)

    Nielsen, L.B.; Nielsen, Henrik Hauch

    2001-01-01

    hamatus) and trout ovary (Oncorhynchus mykiss). Digestion of the P-chain of oxidized insulin resulted in preferential cleavage at Leu(15)-Tyr(16), (47%), Tyr(16)-Leu(17) (34%) and Ala(14)- Leu(15) (18%). Incubation with myofibrils from herring muscle at pH 4.23 showed that the enzyme mainly degraded......Cathepsin D was purified and concentrated 469-fold from a homogenate of Clupea harengus muscle. The purified enzyme is a monomer with a molecular weight of 38 000-39 000. It is inhibited by pepstatin and has optimal activity at pH 2.5 with hemoglobin as the substrate. The isoelectric point is at p...

  13. Production of barley endoprotease B2 in Pichia pastoris and its proteolytic activity against native and recombinant hordeins

    DEFF Research Database (Denmark)

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

    2014-01-01

    -terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni2+-affinity chromatography. Purified protein was evaluated by SDS–PAGE, Western blotting and activity assays...... was 60 °C, thermal stability T50 value was 44 °C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12 h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS–PAGE. The intensities of the B...... and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2 h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were...

  14. Cathepsin D immobilized capillary reactors for on-flow screening assays.

    Science.gov (United States)

    Cornelio, Vivian Estevam; de Moraes, Marcela Cristina; Domingues, Vanessa de Cassia; Fernandes, João Batista; da Silva, Maria Fátima das Gracas Fernandes; Cass, Quezia Bezerra; Vieira, Paulo Cezar

    2018-03-20

    The treatment of diseases using enzymes as targets has called for the development of new and reliable methods for screening. The protease cathepsin D is one such target involved in several diseases such as tumors, degenerative processes, and vital processes of parasites causing schistosomiasis. Herein, we describe the preparation of a fused silica capillary, cathepsin D (CatD)-immobilized enzyme reactor (IMER) using in a multidimensional High Performance Liquid Chromatography-based method (2D-HPLC) and zonal affinity chromatography as an alternative in the search for new ligands. The activity and kinetic parameters of CatD-IMER were evaluated by monitoring the product MOCAc-Gly-Lys-Pro-Ile-Leu-Phe (P-MOCAc) (K M  = 81.9 ± 7.49 μmol/L) generated by cleavage of the fluorogenic substrate MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys(DNP)-d-Arg-NH2 (S-MOCAc). Stability studies have indicated that CatD-IMER retained 20% of activity after 5 months, a relevant result, because proteases are susceptible to autoproteolysis in solution assays with free enzyme. In the search for inhibitors, 12 crude natural product extracts were analyzed using CatD-IMER as the target, resulting in the isolation of different classes of natural products. In addition, 26 compounds obtained from different species of plants were also screened, demonstrating the efficiency and reproducibility of the herein reported assay even in the case of complex matrices such as plant crude extracts. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Expression of recombinant staphylokinase, a fibrin-specific plasminogen activator of bacterial origin, in potato (Solanum tuberosum L.) plants.

    Science.gov (United States)

    Gerszberg, Aneta; Wiktorek-Smagur, Aneta; Hnatuszko-Konka, Katarzyna; Łuchniak, Piotr; Kononowicz, Andrzej K

    2012-03-01

    One of the most dynamically developing sectors of green biotechnology is molecular farming using transgenic plants as natural bioreactors for the large scale production of recombinant proteins with biopharmaceutical and therapeutic values. Such properties are characteristic of certain proteins of bacterial origin, including staphylokinase. For many years, work has been carried out on the use of this protein in thrombolytic therapy. In this study, transgenic Solanum tuberosum plants expressing a CaMV::sak-mgpf-gusA gene fusion, were obtained. AGL1 A. tumefaciens strain was used in the process of transformation. The presence of the staphylokinase gene was confirmed by PCR in 22.5% of the investigated plants. The expression of the fusion transgene was detected using the β-glucuronidase activity assay in 32 putative transgenic plants. Furthermore, on the basis of the GUS histochemical reaction, the transgene expression pattern had a strong, constitutive character in seven of the transformants. The polyacrylamide gel electrophoresis of a protein extract from the SAK/PCR-positive plants, revealed the presence of a119 kDa protein that corresponds to that of the fusion protein SAK-mGFP-GUSA. Western blot analysis, using an antibody against staphylokinase, showed the presence of the staphylokinase domain in the 119 kDa protein in six analyzed transformants. However, the enzymatic test revealed amidolytic activity characteristic of staphylokinase in the protein extract of only one plant. This is the first report on a Solanum tuberosum plant producing a recombinant staphylokinase protein, a plasminogen activator of bacterial origin.

  16. Recombinant mouse PAP has pH-dependent ectonucleotidase activity and acts through A(1-adenosine receptors to mediate antinociception.

    Directory of Open Access Journals (Sweden)

    Nathaniel A Sowa

    Full Text Available Prostatic acid phosphatase (PAP is expressed in nociceptive neurons and functions as an ectonucleotidase. When injected intraspinally, the secretory isoforms of human and bovine PAP protein have potent and long-lasting antinociceptive effects that are dependent on A(1-adenosine receptor (A(1R activation. In this study, we purified the secretory isoform of mouse (mPAP using the baculovirus expression system to determine if recombinant mPAP also had antinociceptive properties. We found that mPAP dephosphorylated AMP, and to a much lesser extent, ADP at neutral pH (pH 7.0. In contrast, mPAP dephosphorylated all purine nucleotides (AMP, ADP, ATP at an acidic pH (pH 5.6. The transmembrane isoform of mPAP had similar pH-dependent ectonucleotidase activity. A single intraspinal injection of mPAP protein had long-lasting (three day antinociceptive properties, including antihyperalgesic and antiallodynic effects in the Complete Freund's Adjuvant (CFA inflammatory pain model. These antinociceptive effects were transiently blocked by the A(1R antagonist 8-cyclopentyl-1, 3-dipropylxanthine (CPX, suggesting mPAP dephosphorylates nucleotides to adenosine to mediate antinociception just like human and bovine PAP. Our studies indicate that PAP has species-conserved antinociceptive effects and has pH-dependent ectonucleotidase activity. The ability to metabolize nucleotides in a pH-dependent manner could be relevant to conditions like inflammation where tissue acidosis and nucleotide release occur. Lastly, our studies demonstrate that recombinant PAP protein can be used to treat chronic pain in animal models.

  17. Cultured Mast Cells from Patients with Asthma and Controls Respond with Similar Sensitivity to Recombinant Der P2-Induced, IgE-Mediated Activation

    DEFF Research Database (Denmark)

    Krohn, I K; Sverrild, A; Lund, G

    2013-01-01

    for mite allergen Der p2. The sensitivity of IgE-mediated activation of mast cells was investigated as FcεRI-mediated upregulation of CD63. Ten subjects were atopic, defined as a positive skin prick test (>3 mm) to at least one of ten common allergens. After activation with recombinant Der p2, the maximum...

  18. Application of synchrotron-radiation-based x-ray microprobe techniques for the analysis of recombination activity of metals precipitated at Si/SiGe misfit dislocations

    CERN Document Server

    Vyvenko, O F; Istratov, A A; Weber, E R; Kittler, M; Seifert, W

    2002-01-01

    In this study we report application of synchrotron-radiation-based x-ray microprobe techniques (the x-ray-beam-induced current (XBIC) and x-ray fluorescence (mu-XRF) methods) to the analysis of the recombination activity and space distribution of copper and iron in the vicinity of dislocations in silicon/silicon-germanium structures. A combination of these two techniques enables one to study the chemical nature of the defects and impurities and their recombination activity in situ and to map metal clusters with a micron-scale resolution. XRF analysis revealed that copper formed clearly distinguishable precipitates along the misfit dislocations. A proportional dependence between the XBIC contrast and the number of copper atoms in the precipitates was established. In hydrogen-passivated iron-contaminated samples we observed clusters of iron precipitates which had no recombination activity detectable by the XBIC technique as well as iron clusters which were not completely passivated.

  19. Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

    Energy Technology Data Exchange (ETDEWEB)

    Nguyen, Minh Vu Chuong, E-mail: mvchuong@yahoo.fr [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Zhang, Leilei [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France); Lhomme, Stanislas; Mouz, Nicolas [PX' Therapeutics, MINATEC/Batiment de Haute Technologie, Grenoble (France); Lenormand, Jean-Luc [HumProTher Laboratory, TheReX/TIMC-IMAG UMR 5525 CNRS UJF, Universite Joseph Fourier, UFR de Medecine, Domaine de la Merci, 38706 La Tronche (France); Lardy, Bernard; Morel, Francoise [GREPI AGIM FRE 3405 CNRS-UJF, Universite Joseph Fourier, Grenoble (France)

    2012-03-16

    Highlights: Black-Right-Pointing-Pointer A comparison of two bacterial cell and cell-free protein expression systems is presented. Black-Right-Pointing-Pointer Soluble and active truncated Nox4 proteins are produced. Black-Right-Pointing-Pointer Nox4 has a constitutive diaphorase activity which is independent of cytosolic factors. Black-Right-Pointing-Pointer Isoform Nox4B is unable to initiate the first electronic transfer step. Black-Right-Pointing-Pointer Findings contribute to the understanding of the mechanism of Nox4 oxidase activity. -- Abstract: The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4 {+-} 1.7 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5 {+-} 2.8 nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26 {+-} 2.6 nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48 {+-} 20.2 nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the

  20. Anti-tumor efficacy of lymphokine-activated killer cells and recombinant interleukin 2 in vivo

    International Nuclear Information System (INIS)

    Mule, J.J.; Shu, S.; Rosenberg, S.A.

    1985-01-01

    The authors showed previously that adoptive immunotherapy with the combination of LAK cells and recombinant IL 2 (RIL 2) can markedly reduce pulmonary micrometastases from multiple sarcomas established 3 days after the i.v. injection of syngeneic tumor cells in C57BL/6 mice. In this report, they analyzed the factors required for successful therapy. Titration analysis in vivo revealed an inverse relationship between the number of pulmonary metastases remaining after treatment and both the number of LAK cells and the amount of RIL 2 administered. Fresh or unstimulated splenocytes had no anti-tumor effect; a 2- to 3-day incubation of splenocytes in RIL 2 was required. LAK cells generated from allogeneic DBA (H-2d) splenocytes were as effective in vivo as syngeneic, C57BL/6 (H-2b) LAK cells. The anti-metastatic capacity of LAK cells was significantly reduced or eliminated when irradiated with 3000 rad before adoptive transfer. The combined therapy of LAK cells plus RIL 2 was shown to be highly effective in mice immunosuppressed by 500 rad total body irradiation and in treating macrometastases established in the lung 10 days after the i.v. injection of sarcoma cells. Further, reduction of both micrometastases and macrometastases could also be achieved by RIL 2 alone when administered at higher levels than were required with LAK cells. The value of LAK cell transfer and of IL 2 administration for the treatment of tumors established at other sites is currently under investigation

  1. Immunoregulatory activities of human immunodeficiency virus (HIV) proteins: Effect of HIV recombinant and synthetic peptides on immunoglobulin synthesis and proliferative responses by normal lymphocytes

    International Nuclear Information System (INIS)

    Nair, M.P.N.; Pottathil, R.; Heimer, E.P.; Schwartz, S.A.

    1988-01-01

    Recombinant and synthetic peptides corresponding to envelope proteins of the human immunodeficiency virus (HIV) were examined for their effects on the activities of lymphocytes from normal donors in vitro. Although lymphocytes cultured with env-gag peptides produced significant amounts of IgG, addition of env-gag peptides to a pokeweed mitogen-induced B-cell activation system resulted in suppression of immunoglobulin synthesis by normal lymphocytes. Recombinant antigens, env-gag and env-80 dihydrofolate reductase (DHFR), produced a substantial proliferative response by peripheral blood mononuclear cells (PBMC) as determined by [ 3 H]thymidine incorporation. PBMC precultured with HIV synthetic peptide env 578-608 also manifested significant proliferative responses as compared to control cultures. CD3 + lymphocytes precultured with recombinant HIV antigens, env-gag and env-80 DHFR, and synthetic HIV peptide, env 487-511, showed moderate but significant proliferative responses. Both recombinant antigens and synthetic peptides also produced a dose-dependent stimulatory effect on proliferation by CD3 - lymphocytes. These studies demonstrate that recombinant and synthetic peptides of the HIV genome express immunoregulatory T- and B-cell epitopes. Identification of unique HIV epitopes with immunogenic and immunoregulatory activities is necessary for the development of an effective vaccine against HIV infection

  2. Activity in mice of recombinant BCG-EgG1Y162 vaccine for Echinococcus granulosus infection.

    Science.gov (United States)

    Ma, Xiumin; Zhao, Hui; Zhang, Fengbo; Zhu, Yuejie; Peng, Shanshan; Ma, Haimei; Cao, Chunbao; Xin, Yan; Yimiti, Delixiati; Wen, Hao; Ding, Jianbing

    2016-01-01

    Cystic hydatid disease is a zoonotic parasitic disease caused by Echinococcus granulosus which is distributed worldwide. The disease is difficult to treat with surgery removal is the only cure treatment. In the high endemic areas, vaccination of humans is believed a way to protect communities from the disease. In this study we vaccinated BALB/c mice with rBCG-EgG1Y162, and then detected the level of IgG and IgE specifically against the recombinant protein by ELISA, rBCG-EgG1Y162 induced strong and specific cellular and humoral immune responses. In vitro study showed that rBCG-EgG1Y162 vaccine not only promote splenocytes proliferation but also active T cell. In addition, the rBCG-EgG1Y162 induced a protection in the mice against secondary infection of Echinococcus granulosus.

  3. Cryopreserved recombinant tissue plasminogen activator for the restoration of occluded central venous access devices in pediatric oncology patients

    International Nuclear Information System (INIS)

    Iqbal, Y.; Al-Katheri, A.; Al-Sedairy, R.; Al-Omari, A.; Abdullah, Mohammed F.; Crankson, S.

    2002-01-01

    Thrombolytic therapy with urokinase 5000 units has been the standard therapy for restoration of thrombosed central catheters. However, with the decreased availability of urokinase, alternatives needed to be sought. The aim of the study was to determine the efficacy, bioactivity, dwell time and cost of cryopreserved recombinant tissue plasminogen activator (rTPA) in the restoration of occluded central venous access devices. For children 10kg, a dose of 1 mg was used. The dwell time was 1-2 hours. Of the 40 courses of rTPA, 39 fully restored central venous line patency (97%). Successful courses were instilled for an average of 1 hour. Cryopreserved rTPA appears to be safe and effective in the dose used to restore the patency of occluded central venous access devices in pediatric oncology patients. (author)

  4. A comparison between recombinant activated factor VII (Aryoseven) and Novoseven in patients with congenital factor VII deficiency.

    Science.gov (United States)

    Faranoush, M; Abolghasemi, Hassan; Toogeh, Gh; Karimi, M; Eshghi, P; Managhchi, M; Hoorfar, H; Dehdezi, B Keikhaei; Mehrvar, A; Khoeiny, B; Kamyar, K; Heshmat, R; Baghaeipour, M R; Mirbehbahani, N B; Fayazfar, R; Ahmadinejad, M; Naderi, M

    2015-11-01

    In order to establish the efficacy and biosimilar nature of AryoSeven to NovoSeven in the treatment of congenital factor VII (FVII) deficiency, patients received either agent at 30 μg/kg, intravenously per week for 4 weeks, in a randomized fashion. The primary aim was to compare FVII:coagulation activity (FVII:C), 20 minutes after recombinant activated FVII (rFVIIa) injection, in the 2 groups. A secondary measure was self-reported bleeding. The median interquartile baseline range of the plasma level of activated FVII (FVIIa) activity in the 2 groups was 1.6 (1.1-14.0) IU/dL and 5.0 (1.1-25.5) IU/dL. All patients achieved levels of FVIIa (FVII:C) >30 IU/dL, 20 minutes after the injection of rFVIIa. Bleeding was similar between the 2 groups, with a comparable decrease in severity and frequency compared to the last month prior to treatment. AryoSeven is similar to NovoSeven in increasing postinjection FVIIa activity as well as in clinical safety and efficacy. © The Author(s) 2014.

  5. EFFECT OF RECOMBINANT TISSUE-PLASMINOGEN ACTIVATOR ON INTRAABDOMINAL ABSCESS FORMATION IN RATS WITH GENERALIZED PERITONITIS

    NARCIS (Netherlands)

    van Goor, Harry; de Graaf, JS; Kooi, K; Sluiter, WJ; Bom, VJJ; van der Meer, J; Bleichrodt, RP

    1994-01-01

    BACKGROUND: During generalized peritonitis, intraabdominal fibrin deposition is stimulated whereas fibrinolytic activity is reduced, which predisposes intra-abdominal abscess formation. We investigated the effects of increasing the intra-abdominal fibrinolytic activity on abscess formation by

  6. Impact of cysteine variants on the structure, activity, and stability of recombinant human α-galactosidase A

    Science.gov (United States)

    Qiu, Huawei; Honey, Denise M; Kingsbury, Jonathan S; Park, Anna; Boudanova, Ekaterina; Wei, Ronnie R; Pan, Clark Q; Edmunds, Tim

    2015-01-01

    Recombinant human α-galactosidase A (rhαGal) is a homodimeric glycoprotein deficient in Fabry disease, a lysosomal storage disorder. In this study, each cysteine residue in rhαGal was replaced with serine to understand the role each cysteine plays in the enzyme structure, function, and stability. Conditioned media from transfected HEK293 cells were assayed for rhαGal expression and enzymatic activity. Activity was only detected in the wild type control and in mutants substituting the free cysteine residues (C90S, C174S, and the C90S/C174S). Cysteine-to-serine substitutions at the other sites lead to the loss of expression and/or activity, consistent with their involvement in the disulfide bonds found in the crystal structure. Purification and further characterization confirmed that the C90S, C174S, and the C90S/C174S mutants are enzymatically active, structurally intact and thermodynamically stable as measured by circular dichroism and thermal denaturation. The purified inactive C142S mutant appeared to have lost part of its alpha-helix secondary structure and had a lower apparent melting temperature. Saturation mutagenesis study on Cys90 and Cys174 resulted in partial loss of activity for Cys174 mutants but multiple mutants at Cys90 with up to 87% higher enzymatic activity (C90T) compared to wild type, suggesting that the two free cysteines play differential roles and that the activity of the enzyme can be modulated by side chain interactions of the free Cys residues. These results enhanced our understanding of rhαGal structure and function, particularly the critical roles that cysteines play in structure, stability, and enzymatic activity. PMID:26044846

  7. 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.

    Science.gov (United States)

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

    2013-11-01

    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.

  8. Recombinant deamidated mutants of Erwinia chrysanthemi L-asparaginase have similar or increased activity compared to wild-type enzyme.

    Science.gov (United States)

    Gervais, David; Foote, Nicholas

    2014-10-01

    The enzyme Erwinia chrysanthemi L-asparaginase (ErA) is an important biopharmaceutical product used in the treatment of acute lymphoblastic leukaemia. Like all proteins, certain asparagine (Asn) residues of ErA are susceptible to deamidation to aspartic acid (Asp), which may be a concern with respect to enzyme activity and potentially to pharmaceutical efficacy. Recombinant ErA mutants containing Asn to Asp changes were expressed, purified and characterised. Two mutants with single deamidation sites (N41D and N281D) were found to have approximately the same specific activity (1,062 and 924 U/mg, respectively) as the wild-type (908 U/mg). However, a double mutant (N41D N281D) had an increased specific activity (1261 U/mg). The N41D mutation conferred a slight increase in the catalytic constant (k cat 657 s(-1)) when compared to the WT (k cat 565 s(-1)), which was further increased in the double mutant, with a k cat of 798 s(-1). Structural analyses showed that the slight changes caused by point mutation of Asn41 to Asp may have reduced the number of hydrogen bonds in this α-helical part of the protein structure, resulting in subtle changes in enzyme turnover, both structurally and catalytically. The increased α-helical content observed with the N41D mutation by circular dichroism spectroscopy correlates with the difference in k cat, but not K m. The N281D mutation resulted in a lower glutaminase activity compared with WT and the N41D mutant, however the N281D mutation also imparted less stability to the enzyme at elevated temperatures. Taken as a whole, these data suggest that ErA deamidation at the Asn41 and Asn281 sites does not affect enzyme activity and should not be a concern during processing, storage or clinical use. The production of recombinant deamidated variants has proven an effective and powerful means of studying the effect of these changes and may be a useful strategy for other biopharmaceutical products.

  9. Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity

    International Nuclear Information System (INIS)

    Cai Xuwei; Fu Xiaolong; Yang Jian; Song Houyan

    2005-01-01

    Objective: To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods: Based on Adeno- X TM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then sub-cloned into the Adeno-X TM genome, which was transformed into E. coli to get recombinant Adeno-X TM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated , and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results: Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0 x 10 11 TCID 50 /ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions: With the aid of Adeno-X TM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose

  10. 78 FR 12074 - Office of Biotechnology Activities; Recombinant DNA Research: Actions Under the NIH Guidelines...

    Science.gov (United States)

    2013-02-21

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology... recommendations of the RAC, the NIH Office of Biotechnology Activities (OBA) concluded that more specific guidance... address or by fax at 301-496-9839 or by mail to the Office of Biotechnology Activities, National...

  11. 76 FR 62816 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Science.gov (United States)

    2011-10-11

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology.... SUMMARY: The Office of Biotechnology Activities (OBA) is updating Appendix B of the NIH Guidelines to... Biotechnology Activities, National Institutes of Health. [FR Doc. 2011-26224 Filed 10-7-11; 8:45 am] BILLING...

  12. 76 FR 3150 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Science.gov (United States)

    2011-01-19

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology... rodent). On July 20, 2010 the NIH Office of Biotechnology Activities (OBA) published a proposed action... Biotechnology Activities, National Institutes of Health, 6705 Rockledge Drive, Suite 750, MSC 7985, Bethesda...

  13. 75 FR 28811 - Office of Biotechnology Activities; Recombinant DNA Research: Proposed Actions Under the NIH...

    Science.gov (United States)

    2010-05-24

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology... Yersinia pestis has been submitted to the NIH Office of Biotechnology Activities (OBA) by the Institutional... Biotechnology Activities, National Institutes of Health. [FR Doc. 2010-12453 Filed 5-21-10; 8:45 am] BILLING...

  14. Activation of Recombinantly Expressed l-Amino Acid Oxidase from Rhizoctonia solani by Sodium Dodecyl Sulfate

    Directory of Open Access Journals (Sweden)

    Katharina Hahn

    2017-12-01

    Full Text Available l-Amino acid oxidases (l-AAO catalyze the oxidative deamination of l-amino acids to the corresponding α-keto acids. The non-covalently bound cofactor FAD is reoxidized by oxygen under formation of hydrogen peroxide. We expressed an active l-AAO from the fungus Rhizoctonia solani as a fusion protein in E. coli. Treatment with small amounts of the detergent sodium dodecyl sulfate (SDS stimulated the activity of the enzyme strongly. Here, we investigated whether other detergents and amphiphilic molecules activate 9His-rsLAAO1. We found that 9His-rsLAAO1 was also activated by sodium tetradecyl sulfate. Other detergents and fatty acids were not effective. Moreover, effects of SDS on the oligomerization state and the protein structure were analyzed. Native and SDS-activated 9His-rsLAAO1 behaved as dimers by size-exclusion chromatography. SDS treatment induced an increase in hydrodynamic radius as observed by size-exclusion chromatography and dynamic light scattering. The activated enzyme showed accelerated thermal inactivation and an exposure of additional protease sites. Changes in tryptophan fluorescence point to a more hydrophilic environment. Moreover, FAD fluorescence increased and a lower concentration of sulfites was sufficient to form adducts with FAD. Taken together, these data point towards a more open conformation of SDS-activated l-amino acid oxidase facilitating access to the active site.

  15. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation.

    Science.gov (United States)

    Balamurugan, Appakalai N; Green, Michael L; Breite, Andrew G; Loganathan, Gopalakrishnan; Wilhelm, Joshua J; Tweed, Benjamin; Vargova, Lenka; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G; Williams, Stuart K; Hering, Bernhard J; Dwulet, Francis E; McCarthy, Robert C

    2016-01-01

    Isolation following a good manufacturing practice-compliant, human islet product requires development of a robust islet isolation procedure where effective limits of key reagents are known. The enzymes used for islet isolation are critical but little is known about the doses of class I and class II collagenase required for successful islet isolation. We used a factorial approach to evaluate the effect of high and low target activities of recombinant class I (rC1) and class II (rC2) collagenase on human islet yield. Consequently, 4 different enzyme formulations with divergent C1:C2 collagenase mass ratios were assessed, each supplemented with the same dose of neutral protease. Both split pancreas and whole pancreas models were used to test enzyme targets (n = 20). Islet yield/g pancreas was compared with historical enzymes (n = 42). Varying the Wunsch (rC2) and collagen degradation activity (CDA, rC1) target dose, and consequently the C1:C2 mass ratio, had no significant effect on tissue digestion. Digestions using higher doses of Wunsch and CDA resulted in comparable islet yields to those obtained with 60% and 50% of those activities, respectively. Factorial analysis revealed no significant main effect of Wunsch activity or CDA for any parameter measured. Aggregate results from 4 different collagenase formulations gave 44% higher islet yield (>5000 islet equivalents/g) in the body/tail of the pancreas (n = 12) when compared with those from the same segment using a standard natural collagenase/protease mixture (n = 6). Additionally, islet yields greater than 5000 islet equivalents/g pancreas were also obtained in whole human pancreas. A broader C1:C2 ratio can be used for human islet isolation than has been used in the past. Recombinant collagenase is an effective replacement for the natural enzyme and we have determined that high islet yield can be obtained even with low doses of rC1:rC2, which is beneficial for the survival of islets.

  16. Endogenous and recombinant type I interferons and disease activity in multiple sclerosis

    DEFF Research Database (Denmark)

    Sellebjerg, Finn; Krakauer, Martin; Limborg, Signe

    2012-01-01

    the percentage of CD4+ T cells expressing CD71 and HLA-DR (activated T cells), and this was associated with an increased risk of clinical disease activity. In contrast, induction of CD71 and HLA-DR was not observed in untreated MS patients with evidence of endogenous type IFN I activity. In conclusion......Although treatment of multiple sclerosis (MS) with the type I interferon (IFN) IFN-ß lowers disease activity, the role of endogenous type I IFN in MS remains controversial. We studied CD4+ T cells and CD4+ T cell subsets, monocytes and dendritic cells by flow cytometry and analysed the relationship...... with endogenous type I IFN-like activity, the effect of IFN-ß therapy, and clinical and magnetic resonance imaging (MRI) disease activity in MS patients. Endogenous type I IFN activity was associated with decreased expression of the integrin subunit CD49d (VLA-4) on CD4+CD26(high) T cells (Th1 helper cells...

  17. Potentiation of apoptosis by histone deacetylase inhibitors and doxorubicin combination: cytoplasmic cathepsin B as a mediator of apoptosis in multiple myeloma.

    Science.gov (United States)

    Cheriyath, V; Kuhns, M A; Kalaycio, M E; Borden, E C

    2011-03-15

    Although inhibitors of histone deacetylase inhibitors (HDACis) in combination with genotoxins potentiate apoptosis, the role of proteases other than caspases in this process remained elusive. Therefore, we examined the potentiation of apoptosis and related mechanisms of HDACis and doxorubicin combination in a panel of myeloma cell lines and in 25 primary myelomas. At IC(50) concentrations, sodium butyrate (an HDACi) or doxorubicin alone caused little apoptosis. However, their combination potentiated apoptosis and synergistically reduced the viability of myeloma cells independent of p53 and caspase 3-7 activation. Potentiated apoptosis correlated with nuclear translocation of apoptosis-inducing factor, suggesting the induction of caspase 3- and 7-independent pathways. Consistent with this, butyrate and doxorubicin combination significantly increased the activity of cytoplasmic cathepsin B. Inhibition of cathepsin B either with a small-molecule inhibitor or downregulation with a siRNA reversed butyrate- and doxorubicin-potentiated apoptosis. Finally, ex vivo, clinically relevant concentrations of butyrate or SAHA (suberoylanilide hydroxamic acid, vorinostat, an HDACi in clinical testing) in combination with doxorubicin significantly (Pmediating apoptosis potentiated by HDACi and doxorubicin combinations in myeloma. Our results support a molecular model of lysosomal-mitochondrial crosstalk in HDACi- and doxorubicin-potentiated apoptosis through the activation of cathepsin B.

  18. Molecular chaperone assisted expression systems: obtaining pure soluble and active recombinant proteins for structural and therapeutic purposes

    CSIR Research Space (South Africa)

    Makhoba, XH

    2015-09-01

    Full Text Available For many years recombinant protein production has been at the center of biosciences used for structural and therapeutic purposes. The production of recombinant proteins in foreign host system such as E. coli has been a biggest challenge. This has...

  19. Pharmacodynamics of recombinant activated factor VII and plasma-derived factor VII in a cohort of severe FVII deficient patients.

    Science.gov (United States)

    van Geffen, Mark; Mathijssen, Natascha C J; Holme, Pål A; Laros-van Gorkom, Britta A P; van Kraaij, Marian G J; Masereeuw, Roselinde; Peyvandi, Flora; van Heerde, Waander L

    2013-07-01

    Recombinant activated factor VII (rFVIIa) and plasma-derived factor VII (pdFVII) are used to prevent bleedings in severe FVII deficient patients, despite their short half-lifes. It is suggested that FVII levels of 15-20 IU/dL are sufficient to maintain hemostasis. We analyzed the pharmacodynamic effects of FVII substitution therapy in the Nijmegen Hemostasis Assay (NHA) that simultaneously measures thrombin and plasmin generation. Ten severe FVII deficient patients were treated with 20 μg/kg rFVIIa or 25 IU/kg pdFVII in a cross-over design. Thrombin generation lag-time (TG-LT) was identified as an effect-response parameter. Pharmacodynamic analysis using a maximum effect model showed 50% reduction of the TG-LT effect at ~2 IU/dL FVII activity for both rFVIIa and pdFVII. The FVII activity to obtain TG-LT comparable to the upper limit of normal range in healthy controls (4 min) was given by the effective concentration (ECnormal), showing sufficient hemostasis at 3-4 IU/dL FVII activity. No association was seen between FVII activity and other thrombin or plasmin generation parameters as measured by NHA. In conclusion, 3-4 IU/dL FVII activity seems sufficient to maintain hemostasis in patients with severe FVII deficiency during prophylaxis. These data may suggest a potential value for measurement of TG-LT in the monitoring of FVII(a) therapy. Copyright © 2013 Elsevier Ltd. All rights reserved.

  20. Reasons for the lack of benefit of immediate angioplasty during recombinant tissue plasminogen activator therapy for acute myocardial infarction: a regional wall motion analysis. European Cooperative Study Group

    NARCIS (Netherlands)

    Arnold, A. E.; Serruys, P. W.; Rutsch, W.; Simoons, M. L.; de Bono, D. P.; Tijssen, J. G.; Lubsen, J.; Verstraete, M.

    1991-01-01

    Regional ventricular wall motion analysis utilizing three different methods was performed on predischarge left ventriculograms from 291 of 367 patients enrolled in a randomized trial of single chain recombinant tissue-type plasminogen activator (rt-PA), aspirin and heparin with and without immediate

  1. Increased volume of distribution for recombinant activated factor VII and longer plasma-derived factor VII half-life may explain their long lasting prophylactic effect

    NARCIS (Netherlands)

    Mathijssen, N.C.J.; Masereeuw, R.; Holme, P.A.; Kraaij, M.G.J. van; Laros, B.A.P.; Peyvandi, F.; Heerde, W.L. van

    2013-01-01

    INTRODUCTION: Prophylaxis with plasma-derived or recombinant activated factor VII is beneficial in severe factor VII deficiency. To understand why prophylactic treatment with both products is efficacious, we conducted a pharmacokinetic study. MATERIALS AND METHODS: Ten factor VII deficient patients

  2. Prediction of human pharmacokinetics of activated recombinant factor VII and B-domain truncated factor VIII from animal population pharmacokinetic models of haemophilia

    DEFF Research Database (Denmark)

    Larsen, Malte Selch; Juul, Rasmus Vestergaard; Groth, Andreas Velsing

    2018-01-01

    activated factor VII (rFVIIa) and recombinant factor VIII (rFVIII) in several experimental animal models using population PK modelling, and apply a simulation-based approach to evaluate how well the developed animal population PK models predict human PK. PK models were developed for rFVIIa and r...

  3. Safety and efficacy of recombinant activated factor VII: a randomized placebo-controlled trial in the setting of bleeding after cardiac surgery

    DEFF Research Database (Denmark)

    Gill, Ravi; Herbertson, Mike; Vuylsteke, Alain

    2009-01-01

    BACKGROUND: Blood loss is a common complication of cardiac surgery. Evidence suggests that recombinant activated factor VII (rFVIIa) can decrease intractable bleeding in patients after cardiac surgery. Our objective was to investigate the safety and possible benefits of rFVIIa in patients who bleed...

  4. The biological activity of a recombinantly expressed (His)(6)-tagged peanut allergen (rAra h 1) is unaffected by endotoxin removal

    DEFF Research Database (Denmark)

    Jensen, Louise Bjerremann; Torp, Anna Maria; Andersen, Sven Bode

    2008-01-01

    The application of recombinant (His)(6)-tagged proteins in cell culture assays is associated with problems due to lipopolysaccharide (LPS) contamination. LPS stimulates cells of the immune system, thereby masking antigen-specific activation of T cells. Due to the affinity of LPS for histidine it ...

  5. The immunophenotypic and immunogenotypic B-cell differentiation arrest in bone marrow of RAG-deficient SCID patients corresponds to residual recombination activities of mutated RAG proteins

    NARCIS (Netherlands)

    J.G. Noordzij; S. de Bruin-Versteeg (Sandra); N.S. Verkaik (Nicole); J.M.J.J. Vossen; R. de Groot (Ronald); E. Bernatowska (Ewa); A.W. Langerak (Anton); D.C. van Gent (Dik); J.J.M. van Dongen (Jacques)

    2002-01-01

    textabstractThe protein products of the recombination activating genes (RAG1 and RAG2) initiate the formation of immunoglobulin (Ig) and T-cell receptors, which are essential for B- and T-cell development, respectively. Mutations in the RAG genes result in severe combined

  6. Investigation of the recombination losses in a three-electrode cylindrical ionization chamber developed for gamma ray dosimetry of fission product activity

    International Nuclear Information System (INIS)

    Ahmad, N.; Matiullah

    1995-01-01

    A three-electrode ionization chamber has been designed and developed for the gamma ray dosimetry of fission product activity and reported elsewhere. In this paper, the (I, V) characteristics of the chamber filled, with argon gas at 1.24 MPa (180 psi) pressure, for fission product gamma rays from spent fuel have been studied. To do so, the chamber was irradiated with gamma rays using different numbers of (i.e. up to 4) spent fuel elements. The plateau region is reached above 1200 V and the detector operating voltage is found to be 2 kV. It is observed that in the plateau region the slope increases with an increase in the exposure rate. The (1/I, 1/V) and (I, 1/V 2 ) characteristic curves reveal the presence of the initial and volume recombination losses. The volume recombination losses are found to be smaller than the initial recombination losses. Both these losses increase with the increasing exposure rate but the increase in the volume recombination losses is slightly greater than that of the initial recombination losses. (orig.)

  7. Neurotherapeutic activity of the recombinant heat shock protein Hsp70 in a model of focal cerebral ischemia in rats

    Directory of Open Access Journals (Sweden)

    Shevtsov MA

    2014-05-01

    Full Text Available Maxim A Shevtsov,1,2 Boris P Nikolaev,3 Ludmila Y Yakovleva,3 Anatolii V Dobrodumov,4 Anastasiy S Dayneko,5 Alexey A Shmonin,5,6 Timur D Vlasov,5 Elena V Melnikova,5 Alexander D Vilisov,4,5 Irina V Guzhova,1 Alexander M Ischenko,3 Anastasiya L Mikhrina,7 Oleg V Galibin,5 Igor V Yakovenko,2 Boris A Margulis1 1Institute of Cytology of the Russian Academy of Sciences (RAS, St Petersburg, Russia; 2AL Polenov Russian Research Scientific Institute of Neurosurgery, St Petersburg, Russia; 3Research Institute of Highly Pure Biopreparations, St Petersburg, Russia; 4Institute of Macromolecular Compounds of the Russian Academy of Sciences (RAS, St Petersburg, Russia; 5First St Petersburg IP Pavlov State Medical University, St Petersburg, Russia; 6Federal Almazov Medical Research Centre, St Petersburg, Russia; 7IM Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences (RAS, St Petersburg, Russia Abstract: Recombinant 70 kDa heat shock protein (Hsp70 is an antiapoptotic protein that has a cell protective activity in stress stimuli and thus could be a useful therapeutic agent in the management of patients with acute ischemic stroke. The neuroprotective and neurotherapeutic activity of recombinant Hsp70 was explored in a model of experimental stroke in rats. Ischemia was produced by the occlusion of the middle cerebral artery for 45 minutes. To assess its neuroprotective capacity, Hsp70, at various concentrations, was intravenously injected 20 minutes prior to ischemia. Forty-eight hours after ischemia, rats were sacrificed and brain tissue sections were stained with 2% triphenyl tetrazolium chloride. Preliminary treatment with Hsp70 significantly reduced the ischemic zone (optimal response at 2.5 mg/kg. To assess Hsp70’s neurotherapeutic activity, we intravenously administered Hsp70 via the tail vein 2 hours after reperfusion (2 hours and 45 minutes after ischemia. Rats were then kept alive for 72 hours. The

  8. Activities of wildtype and mutant p53 in suppression of homologous recombination as measured by a retroviral vector system

    International Nuclear Information System (INIS)

    Lu Xiongbin; Lozano, Guillermina; Donehower, Lawrence A.

    2003-01-01

    DNA repair of double strand breaks, interstrand DNA cross-links, and other types of DNA damage utilizes the processes of homologous recombination and non-homologous end joining to repair the damage. Aberrant homologous recombination is likely to be responsible for a significant fraction of chromosomal deletions, duplications, and translocations that are observed in cancer cells. To facilitate measurement of homologous recombination frequencies in normal cells, mutant cells, and cancer cells, we have developed a high titer retroviral vector containing tandem repeats of mutant versions of a GFP-Zeocin resistance fusion gene and an intact neomycin resistance marker. Recombination between the tandem repeats regenerates a functional GFP-Zeo R marker that can be easily scored. This retroviral vector was used to assess homologous recombination frequencies in human cancer cells and rodent fibroblasts with differing dosages of wild type or mutant p53. Absence of wild type p53 stimulated spontaneous and ionizing radiation-induced homologous recombination, confirming previous studies. Moreover, p53 +/- mouse fibroblasts show elevated levels of homologous recombination compared to their p53 +/+ counterparts following retroviral vector infection, indicating that p53 is haploinsufficient for suppression of homologous recombination. Transfection of vector-containing p53 null Saos-2 cells with various human cancer-associated p53 mutants revealed that these altered p53 proteins retain some recombination suppression function despite being totally inactive for transcriptional transactivation. The retroviral vector utilized in these studies may be useful in performing recombination assays on a wide array of cell types, including those not readily transfected by normal vectors

  9. A role for very low-dose recombinant activated factor VII in refractory bleeding after cardiac surgery: Lessons from an observational study.

    Science.gov (United States)

    Hoffmann, Till; Assmann, Alexander; Dierksen, Angelika; Roussel, Elisabeth; Ullrich, Sebastian; Lichtenberg, Artur; Albert, Alexander; Sixt, Stephan

    2018-04-18

    Although off-label use of recombinant activated factor VII against refractory bleeding is incorporated in current guideline recommendations, safety concerns persist predominantly with respect to thromboembolic complications. We analyzed the safety and efficacy of recombinant activated factor VII at a very low dose in cardiosurgical patients with refractory bleeding. This prospective study includes 1180 cardiosurgical patients at risk of bleeding. Goal-directed substitution was based on real-time laboratory testing and clinical scoring of the bleeding intensity. All patients who fulfilled the criteria for enhanced risk of bleeding (n = 281) were consequently included in the present analysis. Patients in whom refractory bleeding developed despite substitution with specific hemostatic compounds (n = 167) received a single shot of very low-dose recombinant activated factor VII (≤20 μg/kg). Mortality and risk of thromboembolic complications, and freedom from stroke and acute myocardial infarction in particular, were analyzed (vs patients without recombinant activated factor VII) by multivariable logistic and Cox regression analyses, as well as Kaplan-Meier estimates. There was no increase in rates of mortality (30-day mortality 4.2% vs 7.0% with P = .418; follow-up survival 85.6% at 13.0 [interquartile range, 8.4-15.7] months vs 80.7% at 10.2 [interquartile range, 7.2-16.1] months with P = .151), thromboembolic complications (6.6% vs 9.6% with P = .637), renal insufficiency, need for percutaneous coronary intervention, duration of ventilation, duration of hospital stay, or rehospitalization in patients receiving very low-dose recombinant activated factor VII compared with patients not receiving recombinant activated factor VII. Complete hemostasis without any need for further hemostatic treatment was achieved after very low-dose recombinant activated factor VII administration in the majority of patients (up to 88.6% vs 0% with P factor VII treatment of

  10. Mediator facilitates transcriptional activation and dynamic long-range contacts at the IgH locus during class switch recombination.

    Science.gov (United States)

    Thomas-Claudepierre, Anne-Sophie; Robert, Isabelle; Rocha, Pedro P; Raviram, Ramya; Schiavo, Ebe; Heyer, Vincent; Bonneau, Richard; Luo, Vincent M; Reddy, Janardan K; Borggrefe, Tilman; Skok, Jane A; Reina-San-Martin, Bernardo

    2016-03-07

    Immunoglobulin (Ig) class switch recombination (CSR) is initiated by the transcription-coupled recruitment of activation-induced cytidine deaminase (AID) to Ig switch regions (S regions). During CSR, the IgH locus undergoes dynamic three-dimensional structural changes in which promoters, enhancers, and S regions are brought to close proximity. Nevertheless, little is known about the underlying mechanisms. In this study, we show that Med1 and Med12, two subunits of the mediator complex implicated in transcription initiation and long-range enhancer/promoter loop formation, are dynamically recruited to the IgH locus enhancers and the acceptor regions during CSR and that their knockdown in CH12 cells results in impaired CSR. Furthermore, we show that conditional inactivation of Med1 in B cells results in defective CSR and reduced acceptor S region transcription. Finally, we show that in B cells undergoing CSR, the dynamic long-range contacts between the IgH enhancers and the acceptor regions correlate with Med1 and Med12 binding and that they happen at a reduced frequency in Med1-deficient B cells. Our results implicate the mediator complex in the mechanism of CSR and are consistent with a model in which mediator facilitates the long-range contacts between S regions and the IgH locus enhancers during CSR and their transcriptional activation. © 2016 Thomas-Claudepierre et al.

  11. Characterization of the secreted cathepsin B cysteine proteases family of the carcinogenic liver fluke Clonorchis sinensis.

    Science.gov (United States)

    Chen, Wenjun; Wang, Xiaoyun; Lv, Xiaoli; Tian, Yanli; Xu, Yanquan; Mao, Qiang; Shang, Mei; Li, Xuerong; Huang, Yan; Yu, Xinbing

    2014-09-01

    Clonorchis sinensis excretory/secretory products (ESP) have gained high attentions because of their potential to be vaccine candidates and drug targets in C. sinensis prevention. In this study, we extensively profiled the characteristics of four C. sinensis cathepsin B cysteine proteases (CsCB1, CsCB2, CsCB3, and CsCB4). Bioinformatics analysis showed all CsCBs contained signal peptides at the N-terminal. Functional domains and residues were found in CsCB sequences. We expressed four CsCBs and profiled immune responses followed by vaccine trials. Recombinant CsCBs could induce high IgG titers, indicating high immunogenicity of CsCB family. Additionally, ELISA results showed that both IgG1 and IgG2a levels apparently increased post-immunization with all four CsCBs, showing that combined Th1/Th2 immune responses were triggered by CsCB family. Both Real-time polymerase chain reaction (RT-PCR) and Western blotting confirmed that four CsCBs have distinct expression patterns in C. sinensis life stages. More importantly, we validated our hypothesis that CsCBs were C. sinensis excretory/secretory products. CsCBs could be recognized by C. sinensis-infected sera throughout the infection period, indicating that secreted CsCBs are immune triggers during C. sinensis infection. The protective effect was assessed by comparing the worm burden and egg per gram (EPG) between CsCB group and control group, showing that worm burden (P sinensis excretory/secretory products that may regulate host immune responses.

  12. 75 FR 69687 - Office of Biotechnology Activities Recombinant DNA Research: Proposed Actions Under the NIH...

    Science.gov (United States)

    2010-11-15

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology... to the NIH Office of Biotechnology Activities (OBA). The data to be considered for certifying a new... same e-mail address or by fax at 301-496-9839 or sent by U.S. mail to the Office of Biotechnology...

  13. 76 FR 44339 - Office of Biotechnology Activities; Recombinant DNA Research: Action Under the NIH Guidelines for...

    Science.gov (United States)

    2011-07-25

    ... DEPARTMENT OF HEALTH AND HUMAN SERVICES National Institutes of Health Office of Biotechnology... Services. ACTION: Proposed Minor Action under the NIH Guidelines. SUMMARY: The Office of Biotechnology....nih.gov , telephone (301-496-9838), or mail to the Office of Biotechnology Activities, National...

  14. Neuronal targeting, internalization, and biological activity of a recombinant atoxic derivative of botulinum neurotoxin A

    Science.gov (United States)

    Botulinum neurotoxins (BoNT) have the unique capacity to cross epithelial barriers, target neuromuscular junctions, and translocate active metalloprotease component to the cytosol of motor neurons. We have taken advantage of the molecular carriers responsible for this trafficking to create a family ...

  15. Differential expression of Cathepsin S and X in the spinal cord of a rat neuropathic pain model

    Directory of Open Access Journals (Sweden)

    Schmitz Beate

    2008-08-01

    Full Text Available Abstract Background Ample evidence suggests a substantial contribution of cellular and molecular changes in the spinal cord to the induction and persistence of chronic neuropathic pain conditions. While for a long time, proteases were mainly considered as protein degrading enzymes, they are now receiving growing interest as signalling molecules in the pain pathology. In the present study we focused on two cathepsins, CATS and CATX, and studied their spatiotemporal expression and activity during the development and progression of neuropathic pain in the CNS of the rat 5th lumbar spinal nerve transection model (L5T. Results Immediately after the lesion, both cathepsins, CATS and CATX, were upregulated in the spinal cord. Moreover, we succeeded in measuring the activity of CATX, which was substantially increased after L5T. The differential expression of these proteins exhibited the same spatial distribution and temporal progression in the spinal cord, progressing up to the medulla oblongata in the late phase of chronic pain. The cellular distribution of CATS and CATX was, however, considerably different. Conclusion The cellular distribution and the spatio-temporal development of the altered expression of CATS and CATX suggest that these proteins are important players in the spinal mechanisms involved in chronic pain induction and maintenance.

  16. Structural Dynamics Investigation of Human Family 1 & 2 Cystatin-Cathepsin L1 Interaction: A Comparison of Binding Modes.

    Directory of Open Access Journals (Sweden)

    Suman Kumar Nandy

    Full Text Available Cystatin superfamily is a large group of evolutionarily related proteins involved in numerous physiological activities through their inhibitory activity towards cysteine proteases. Despite sharing the same cystatin fold, and inhibiting cysteine proteases through the same tripartite edge involving highly conserved N-terminal region, L1 and L2 loop; cystatins differ widely in their inhibitory affinity towards C1 family of cysteine proteases and molecular details of these interactions are still elusive. In this study, inhibitory interactions of human family 1 & 2 cystatins with cathepsin L1 are predicted and their stability and viability are verified through protein docking & comparative molecular dynamics. An overall stabilization effect is observed in all cystatins on complex formation. Complexes are mostly dominated by van der Waals interaction but the relative participation of the conserved regions varied extensively. While van der Waals contacts prevail in L1 and L2 loop, N-terminal segment chiefly acts as electrostatic interaction site. In fact the comparative dynamics study points towards the instrumental role of L1 loop in directing the total interaction profile of the complex either towards electrostatic or van der Waals contacts. The key amino acid residues surfaced via interaction energy, hydrogen bonding and solvent accessible surface area analysis for each cystatin-cathepsin L1 complex influence the mode of binding and thus control the diverse inhibitory affinity of cystatins towards cysteine proteases.

  17. The protease degrading sperm histones post-fertilization in sea urchin eggs is a nuclear cathepsin L that is further required for embryo development.

    Directory of Open Access Journals (Sweden)

    Violeta Morin

    Full Text Available Proteolysis of sperm histones in the sea urchin male pronucleus is the consequence of the activation at fertilization of a maternal cysteine protease. We previously showed that this protein is required for male chromatin remodelling and for cell-cycle progression in the newly formed embryos. This enzyme is present in the nucleus of unfertilized eggs and is rapidly recruited to the male pronucleus after insemination. Interestingly, this cysteine-protease remains co-localized with chromatin during S phase of the first cell cycle, migrates to the mitotic spindle in M-phase and is re-located to the nuclei of daughter cells after cytokinesis. Here we identified the protease encoding cDNA and found a high sequence identity to cathepsin proteases of various organisms. A phylogenetical analysis clearly demonstrates that this sperm histone protease (SpHp belongs to the cathepsin L sub-type. After an initial phase of ubiquitous expression throughout cleavage stages, SpHp gene transcripts become restricted to endomesodermic territories during the blastula stage. The transcripts are localized in the invaginating endoderm during gastrulation and a gut specific pattern continues through the prism and early pluteus stages. In addition, a concomitant expression of SpHp transcripts is detected in cells of the skeletogenic lineage and in accordance a pharmacological disruption of SpHp activity prevents growth of skeletal rods. These results further document the role of this nuclear cathepsin L during development.

  18. Azilsartan increases levels of IL-10, down-regulates MMP-2, MMP-9, RANKL/RANK, Cathepsin K and up-regulates OPG in an experimental periodontitis model.

    Directory of Open Access Journals (Sweden)

    Aurigena Antunes de Araújo

    Full Text Available AIMS: The aim of this study was to evaluate the effects of azilsartan (AZT on bone loss, inflammation, and the expression of matrix metallo proteinases (MMPs, receptor activator of nuclear factor κB ligand (RANKL, receptor activator of nuclear factor κB (RANK, osteoprotegerin (OPG, cyclooxygenase-2 (COX-2, and cathepsin K in periodontal tissue in a rat model of ligature-induced periodontitis. MATERIALS AND METHODS: Male Wistar albino rats were randomly divided into 5 groups of 10 rats each: (1 nonligated, water; (2 ligated, water; (3 ligated, 1 mg/kg AZT; (4 ligated, 5 mg/kg AZT; and (5 ligated, 10 mg/kg AZT. All groups were treated with saline or AZT for 10 days. Periodontal tissues were analyzed by histopathology and immunohistochemical detection of MMP-2, MMP-9, COX-2, RANKL, RANK, OPG, and cathepsin K. Levels of IL-1β, IL-10, TNF-α, myeloperoxidase (MPO, and glutathione (GSH were determined by ELISA. RESULTS: Treatment with 5 mg/kg AZT resulted in reduced MPO (p<0.05 and IL-1β (p<0.05, increased levels of IL-10 (p<0.05, and reduced expression of MMP-2, MMP-9, COX-2, RANK, RANKL, cathepsin K, and increased expression of OPG. CONCLUSIONS: These findings reveal that AZT increases anti-inflammatory cytokines and GSH and decreases bone loss in ligature-induced periodontitis in rats.

  19. Mapping Local Cytosolic Enzymatic Activity in Human Esophageal Mucosa with Porous Silicon Nanoneedles.

    Science.gov (United States)

    Chiappini, Ciro; Campagnolo, Paola; Almeida, Carina S; Abbassi-Ghadi, Nima; Chow, Lesley W; Hanna, George B; Stevens, Molly M

    2015-09-16

    Porous silicon nanoneedles can map Cathepsin B activity across normal and tumor human esophageal mucosa. Assembling a peptide-based Cathepsin B cleavable sensor over a large array of nano-needles allows the discrimination of cancer cells from healthy ones in mixed culture. The same sensor applied to tissue can map Cathepsin B activity with high resolution across the tumor margin area of esophageal adenocarcinoma. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  20. Serum and saliva levels of cathepsin L in patients with acute coronary syndrome.

    Science.gov (United States)

    Mirzaii-Dizgah, Iraj; Riahi, Esmail

    2011-03-01

    Coronary artery disease (CAD) is the major cause of death nearly all over the world, and accurate and rapid diagnosis of CAD is of major medical and economic importance. The aim of this study was to evaluate the serum and saliva levels of cathepsin L in patients with acute coronary syndrome (ACS). In a cross-sectional study, 39 patients with ACS and 28 with controls were recruited to the study, and cathepsin L levels were measured in serum, resting saliva, and stimulated saliva obtained 12 and 24 h after the onset of ACS by ELISA method. Statistical analyses of Fisher's exact test, the Student's t-test or Kruskal-Wallis test were performed. Stimulated saliva cathepsin L levels in patients with ACS 12 hours but not 24 hours after admission showed significant decrease compared with that in control subjects. However, there were no significant differences in serum and unstimulated saliva cathepsin L levels between groups. Serum and saliva levels of cathepsin L remain unchanged in patients with ACS and hence may not be a promising factor in CAD risk assessment. It seems that serum and saliva cathepsin L may not be a good biomarker for CHD. CAD: Coronary artery disease, ACS: Acute coronary syndrome, CHD: Coronary heart disease, EU: Emergency unit, MI: Myocardial infarction. Cathepsin L, Acute coronary syndrome, Resting saliva, Stimulated saliva. How to cite this article: Mirzaii-Dizgah I, Riahi E. Serum and Saliva Levels of Cathepsin L in Patients with Acute Coronary Syndrome. J Contemp Dent Pract 2011;12(2):114-119.

  1. Design of Recombinant Stem Cell Factor macrophage Colony Stimulating Factor Fusion Proteins and their Biological Activity In Vitro

    Science.gov (United States)

    Chen, Tao; Yang, Jie; Wang, Yuelang; Zhan, Chenyang; Zang, Yuhui; Qin, Junchuan

    2005-05-01

    Stem cell factor (SCF) and macrophage colony stimulating factor (M-CSF) can act in synergistic way to promote the growth of mononuclear phagocytes. SCF-M-CSF fusion proteins were designed on the computer using the Homology and Biopolymer modules of the software packages InsightII. Several existing crystal structures were used as templates to generate models of the complexes of receptor with fusion protein. The structure rationality of the fusion protein incorporated a series of flexible linker peptide was analyzed on InsightII system. Then, a suitable peptide GGGGSGGGGSGG was chosen for the fusion protein. Two recombinant SCF-M-CSF fusion proteins were generated by construction of a plasmid in which the coding regions of human SCF (1-165aa) and M-CSF (1-149aa) cDNA were connected by this linker peptide coding sequence followed by subsequent expression in insect cell. The results of Western blot and activity analysis showed that these two recombinant fusion proteins existed as a dimer with a molecular weight of 84 KD under non-reducing conditions and a monomer of 42 KD at reducing condition. The results of cell proliferation assays showed that each fusion protein induced a dose-dependent proliferative response. At equimolar concentration, SCF/M-CSF was about 20 times more potent than the standard monomeric SCF in stimulating TF-1 cell line growth, while M-CSF/SCF was 10 times of monomeric SCF. No activity difference of M-CSF/SCF or SCF/M-CSF to M-CSF (at same molar) was found in stimulating the HL-60 cell linear growth. The synergistic effect of SCF and M-CSF moieties in the fusion proteins was demonstrated by the result of clonogenic assay performed with human bone mononuclear, in which both SCF/M-CSF and M-CSF/SCF induced much higher number of CFU-M than equimolar amount of SCF or M-CSF or that of two cytokines mixture.

  2. Biostable Agonists that Match or Exceed Activity of Native Insect Kinins on Recombinant Arthropod GPCRs

    Science.gov (United States)

    2009-01-01

    Drosophila melanogaster and the honey bee Apis mellifera. Prog. Neurobiol. 80, 1–19. Holman, G.M., Nachman, R.J., Wright, M.S., 1990. Insect...Introduction G- Protein -coupled receptors (GPCRs) are seven transmembrane cell surface proteins that are activated by diverse stimuli such as biogenic amines...neuropeptides and protein hormones. A distinct intracellular response, mostly through their heterotrimeric G-pro- tein, is initiated when GPCRs are

  3. Processing of natural and recombinant CXCR3-targeting chemokines and implications for biological activity.

    Science.gov (United States)

    Hensbergen, P J; van der Raaij-Helmer, E M; Dijkman, R; van der Schors, R C; Werner-Felmayer, G; Boorsma, D M; Scheper, R J; Willemze, R; Tensen, C P

    2001-09-01

    Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.

  4. Expression and fast preparation of biologically active recombinant human coagulation factor VII in CHO-K1 cells.

    Science.gov (United States)

    Xiao, W; Li, C Q; Xiao, X P; Lin, F Z

    2013-12-16

    Human coagulation factor VII (FVII) plays an important role in the blood coagulation process and exists in micro amounts in human plasma; therefore, any attempt at the large-scale production of FVII in significant quantities is challenging. The purpose of this study was to express and obtain biologically active recombinant FVII (rFVII) from Chinese hamster ovary K1 (CHO-K1) cells. The full-length FVII cDNA was isolated from a HepG2 cell line and then subcloned in pcDNA3.1 to construct an expression vector, pcDNA-FVII. CHO-K1 cells were transfected with 1 µg pcDNA-FVII. The cell line that stably expressed secretory FVII was screened using 900 µg/mL G418. The FVII copy number in CHO-K1 cells was detected by quantitative polymerase chain reaction (qPCR). The rFVII was purified in ligand affinity chromatography medium. The purified protein was detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis. The biological activity of the purified FVII protein was determined by a prothrombin time assay. Three cell lines that permanently expressed rFVII were screened. The qPCR results demonstrated that each CHO-K1 cell harbored two FVII DNA copies. The SDS-PAGE and Western blot analysis showed that the purified protein was about 50 kDa. The purity of the target protein was 95%. The prothrombin time assay indicated that the FVII-specific activity of rFVII was 2573 ± 75 IU/mg. This method enabled the fast preparation of high-purity rFVII from CHO-K1 cells, and the purified protein had good biological activity.

  5. Integrin specificity and enhanced cellular activities associated with surfaces presenting a recombinant fibronectin fragment compared to RGD supports.

    Science.gov (United States)

    Petrie, Timothy A; Capadona, Jeffrey R; Reyes, Catherine D; García, Andrés J

    2006-11-01

    Biomimetic strategies focusing on presenting short bioadhesive oligopeptides, including the arginine-glycine-aspartic acid (RGD) motif present in numerous adhesive proteins, on a non-fouling support have emerged as promising approaches to improve cellular activities and healing responses. Nevertheless, these bio-inspired strategies are limited by low activity of the oligopeptides compared to the native ligand due to the absence of complementary or modulatory domains. In the present analysis, we generated well-defined biointerfaces presenting RGD-based ligands of increasing complexity to directly compare their biological activities in terms of cell adhesion strength, integrin binding and signaling. Mixed self-assembled monolayers of alkanethiols on gold were optimized to engineer robust supports that present anchoring groups for ligand tethering within a non-fouling, protein adsorption-resistant background. Controlled bioadhesive interfaces were generated by tethering adhesive ligands via standard peptide chemistry. On a molar basis, biointerfaces functionalized with the FNIII7-10 recombinant fragment presenting the RGD and PHSRN adhesive motifs in the correct structural context exhibited significantly higher adhesion strength, FAK activation, and cell proliferation rate than supports presenting RGD ligand or RGD-PHSRN, an oligopeptide presenting these two sites separated by a polyglycine linker. Moreover, FNIII7-10-functionalized surfaces displayed specificity for alpha5beta1 integrin, while cell adhesion to supports presenting RGD or RGD-PHSRN was primarily mediated by alphavbeta3 integrin. These results are significant to the rational engineering of bioactive materials that convey integrin binding specificity for directed cellular and tissue responses in biomedical and biotechnological applications.

  6. Spectrophotometric activity microassay for pure and recombinant cytochrome P450-type nitric oxide reductase

    CSIR Research Space (South Africa)

    Garny, S

    2014-02-01

    Full Text Available spectrophotometric quantification of NADH [19]. Kaya et al. (2004) [19] demonstrated the linearly proportional relationship between the oxidation of NADH to NAD+, with the release of NO from NOC-5, but did not develop a kinetic assay for NOR activity using... this principle, the primary aim of this study. Nakahara et al. (1993) [10] determined the stoichiometry of NO reduction by NOR as 2:1:1 for NO:NADH:N2O. Therefore, for each NADH oxidized to NAD+, two molecules of NO are converted to N2O (refer Equation 1). 2...

  7. Activation of the cellular unfolded protein response by recombinant adeno-associated virus vectors.

    Directory of Open Access Journals (Sweden)

    Balaji Balakrishnan

    Full Text Available The unfolded protein response (UPR is a stress-induced cyto-protective mechanism elicited towards an influx of large amount of proteins in the endoplasmic reticulum (ER. In the present study, we evaluated if AAV manipulates the UPR pathways during its infection. We first examined the role of the three major UPR axes, namely, endoribonuclease inositol-requiring enzyme-1 (IRE1α, activating transcription factor 6 (ATF6 and PKR-like ER kinase (PERK in AAV infected cells. Total RNA from mock or AAV infected HeLa cells were used to determine the levels of 8 different ER-stress responsive transcripts from these pathways. We observed a significant up-regulation of IRE1α (up to 11 fold and PERK (up to 8 fold genes 12-48 hours after infection with self-complementary (scAAV2 but less prominent with single-stranded (ssAAV2 vectors. Further studies demonstrated that scAAV1 and scAAV6 also induce cellular UPR in vitro, with AAV1 vectors activating the PERK pathway (3 fold while AAV6 vectors induced a significant increase on all the three major UPR pathways [6-16 fold]. These data suggest that the type and strength of UPR activation is dependent on the viral capsid. We then examined if transient inhibition of UPR pathways by RNA interference has an effect on AAV transduction. siRNA mediated silencing of PERK and IRE1α had a modest effect on AAV2 and AAV6 mediated gene expression (∼1.5-2 fold in vitro. Furthermore, hepatic gene transfer of scAAV2 vectors in vivo, strongly elevated IRE1α and PERK pathways (2 and 3.5 fold, respectively. However, when animals were pre-treated with a pharmacological UPR inhibitor (metformin during scAAV2 gene transfer, the UPR signalling and its subsequent inflammatory response was attenuated concomitant to a modest 2.8 fold increase in transgene expression. Collectively, these data suggest that AAV vectors activate the cellular UPR pathways and their selective inhibition may be beneficial during AAV mediated gene transfer.

  8. Spectrum Recombination.

    Science.gov (United States)

    Greenslade, Thomas B., Jr.

    1984-01-01

    Describes several methods of executing lecture demonstrations involving the recombination of the spectrum. Groups the techniques into two general classes: bringing selected portions of the spectrum together using lenses or mirrors and blurring the colors by rapid movement or foreshortening. (JM)

  9. In Plant Activation: An Inducible, Hyperexpression Platform for Recombinant Protein Production in Plants[W][OPEN

    Science.gov (United States)

    Dugdale, Benjamin; Mortimer, Cara L.; Kato, Maiko; James, Tess A.; Harding, Robert M.; Dale, James L.

    2013-01-01

    In this study, we describe a novel protein production platform that provides both activation and amplification of transgene expression in planta. The In Plant Activation (INPACT) system is based on the replication machinery of tobacco yellow dwarf mastrevirus (TYDV) and is essentially transient gene expression from a stably transformed plant, thus combining the advantages of both means of expression. The INPACT cassette is uniquely arranged such that the gene of interest is split and only reconstituted in the presence of the TYDV-encoded Rep/RepA proteins. Rep/RepA expression is placed under the control of the AlcA:AlcR gene switch, which is responsive to trace levels of ethanol. Transgenic tobacco (Nicotiana tabacum cv Samsun) plants containing an INPACT cassette encoding the β-glucuronidase (GUS) reporter had negligible background expression but accumulated very high GUS levels (up to 10% total soluble protein) throughout the plant, within 3 d of a 1% ethanol application. The GUS reporter was replaced with a gene encoding a lethal ribonuclease, barnase, demonstrating that the INPACT system provides exquisite control of transgene expression and can be adapted to potentially toxic or inhibitory compounds. The INPACT gene expression platform is scalable, not host-limited, and has been used to express both a therapeutic and an industrial protein. PMID:23839786

  10. Analysis of the Peroxidase Activity of Rice (Oryza Sativa) Recombinant Hemoglobin 1: Implications for the In Vivo Function of Hexacoordinate Non-Symbiotic Hemoglobins in Plants

    Science.gov (United States)

    In plants, it has been proposed that hexacoordinate (class 1) non-symbiotic Hbs (nsHb-1) function in vivo as peroxidases. However, little is known about the peroxidase activity of nsHb-1. We evaluated the peroxidase activity of rice recombinant Hb1 (a nsHb-1) by using the guaiacol/H2O2 system at pH ...

  11. A SEP tag enhances the expression, solubility and yield of recombinant TEV protease without altering its activity.

    Science.gov (United States)

    Nautiyal, Kalpana; Kuroda, Yutaka

    2018-05-25

    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.

  12. Recombinant Trichoderma harzianum endoglucanase I (Cel7B) is a highly acidic and promiscuous carbohydrate-active enzyme.

    Science.gov (United States)

    Pellegrini, Vanessa O A; Serpa, Viviane Isabel; Godoy, Andre S; Camilo, Cesar M; Bernardes, Amanda; Rezende, Camila A; Junior, Nei Pereira; Franco Cairo, João Paulo L; Squina, Fabio M; Polikarpov, Igor

    2015-11-01

    Trichoderma filamentous fungi have been investigated due to their ability to secrete cellulases which find various biotechnological applications such as biomass hydrolysis and cellulosic ethanol production. Previous studies demonstrated that Trichoderma harzianum IOC-3844 has a high degree of cellulolytic activity and potential for biomass hydrolysis. However, enzymatic, biochemical, and structural studies of cellulases from T. harzianum are scarce. This work reports biochemical characterization of the recombinant endoglucanase I from T. harzianum, ThCel7B, and its catalytic core domain. The constructs display optimum activity at 55 °C and a surprisingly acidic pH optimum of 3.0. The full-length enzyme is able to hydrolyze a variety of substrates, with high specific activity: 75 U/mg for β-glucan, 46 U/mg toward xyloglucan, 39 U/mg for lichenan, 26 U/mg for carboxymethyl cellulose, 18 U/mg for 4-nitrophenyl β-D-cellobioside, 16 U/mg for rye arabinoxylan, and 12 U/mg toward xylan. The enzyme also hydrolyzed filter paper, phosphoric acid swollen cellulose, Sigmacell 20, Avicel PH-101, and cellulose, albeit with lower efficiency. The ThCel7B catalytic domain displays similar substrate diversity. Fluorescence-based thermal shift assays showed that thermal stability is highest at pH 5.0. We determined kinetic parameters and analyzed a pattern of oligosaccharide substrates hydrolysis, revealing cellobiose as a final product of C6 degradation. Finally, we visualized effects of ThCel7B on oat spelt using scanning electron microscopy, demonstrating the morphological changes of the substrate during the hydrolysis. The acidic behavior of ThCel7B and its considerable thermostability hold a promise of its industrial applications and other biotechnological uses under extremely acidic conditions.

  13. Optimization of recombinant bacteria expressing dsRNA to enhance insecticidal activity against a lepidopteran insect, Spodoptera exigua.

    Directory of Open Access Journals (Sweden)

    Mohammad Vatanparast

    Full Text Available Double-stranded RNA (dsRNA has been applied to control insect pests due to its induction of RNA interference (RNAi of a specific target gene expression. However, developing dsRNA-based insecticidal agent has been a great challenge especially against lepidopteran insect pests due to variations in RNAi efficiency. The objective of this study was to screen genes of chymotrypsins (SeCHYs essential for the survival of the beet armyworm, Spodoptera exigua, to construct insecticidal dsRNA. In addition, an optimal oral delivery method was developed using recombinant bacteria. At least 7 SeCHY genes were predicted from S. exigua transcriptomes. Subsequent analyses indicated that SeCHY2 was widely expressed in different developmental stages and larval tissues by RT-PCR and its expression knockdown by RNAi caused high mortality along with immunosuppression. However, a large amount of dsRNA was required to efficiently kill late instars of S. exigua because of high RNase activity in their midgut lumen. To minimize dsRNA degradation, bacterial expression and formulation of dsRNA were performed in HT115 Escherichia coli using L4440 expression vector. dsRNA (300 bp specific to SeCHY2 overexpressed in E. coli was toxic to S. exigua larvae after oral administration. To enhance dsRNA release from E. coli, bacterial cells were sonicated before oral administration. RNAi efficiency of sonicated bacteria was significantly increased, causing higher larval mortality at oral administration. Moreover, targeting young larvae possessing weak RNase activity in the midgut lumen significantly enhanced RNAi efficiency and subsequent insecticidal activity against S. exigua.

  14. Role of human recombinant activated protein C and low dose corticosteroid therapy in sepsis

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    Aparna Shukla

    2010-01-01

    Full Text Available Despite advances in modern medicine, sepsis remains a complex syndrome that has been associated with significant morbidity and mortality. Multiple organ failure associated with sepsis leads to high mortality and morbidity. About 28 - 50% deaths have been reported in patients with sepsis. The number of sepsis patients is increasing, with considerable burden on healthcare facilities. Various factors leading to a rise in the incidence of sepsis are (1 Improvement of diagnostic procedures (2 Increase in the number of immunocompromised patients taking treatment for various autoimmune disease, carcinomas, organ transplantation (3 Advances in intensive procedures (4 Nosocomial infections (5 Extensive use of antibiotics. With the better understanding of sepsis various modalities to modify pathophysiological response of septic patients have developed. Activated protein C and low-dose corticosteroid therapy have been tried in patients, with variable results.

  15. Massive Pulmonary Embolism: Treatment with Thrombus Fragmentation and Local Fibrinolysis with Recombinant Human-Tissue Plasminogen Activator

    International Nuclear Information System (INIS)

    Stock, Klaus Wilhelm; Jacob, Augustinus Ludwig; Schnabel, Karl Jakob; Bongartz, Georg; Steinbrich, Wolfgang

    1997-01-01

    Purpose: To report the results of thrombus fragmentation in combination with local fibrinolysis using recombinant human-tissue plasminogen activator (rtPA) in patients with massive pulmonary embolism. Methods: Five patients with massive pulmonary embolism were treated with thrombus fragmentation followed by intrapulmonary injection of rtPA. Clot fragmentation was performed with a guidewire, angiographic catheter, and balloon catheter. Three patients had undergone recent surgery; one of them received a reduced dosage of rtPA. Results: All patients survived and showed clinical improvement with a resultant significant (p < 0.05) decrease in the pulmonary blood pressure (mean systolic pulmonary blood pressure before treatment, 49 mmHg; 4 hr after treatment, 28 mmHg). Angiographic follow-up in three patients revealed a decrease in thrombus material and an increase in pulmonary perfusion. Two patients developed retroperitoneal hematomas requiring transfusion. Conclusion: Clot fragmentation and local fibrinolysis with rtPA was an effective therapy for massive pulmonary embolism. Bleeding at the puncture site was a frequent complication

  16. Outcomes Following Three-Factor Inactive Prothrombin Complex Concentrate Versus Recombinant Activated Factor VII Administration During Cardiac Surgery.

    Science.gov (United States)

    Harper, Patrick C; Smith, Mark M; Brinkman, Nathan J; Passe, Melissa A; Schroeder, Darrell R; Said, Sameh M; Nuttall, Gregory A; Oliver, William C; Barbara, David W

    2018-02-01

    To compare outcomes following inactive prothrombin complex concentrate (PCC) or recombinant activated factor VII (rFVIIa) administration during cardiac surgery. Retrospective propensity-matched analysis. Academic tertiary-care center. Patients undergoing cardiac surgery requiring cardiopulmonary bypass who received either rFVIIa or the inactive 3-factor PCC. Outcomes following intraoperative administration of rFVIIa (263) or factor IX complex (72) as rescue therapy to treat bleeding. In the 24 hours after surgery, propensity-matched patients receiving PCC versus rFVIIa had significantly less chest tube outputs (median difference -464 mL, 95% confidence interval [CI] -819 mL to -110 mL), fresh frozen plasma transfusion rates (17% v 38%, p = 0.028), and platelet transfusion rates (26% v 49%, p = 0.027). There were no significant differences between propensity-matched groups in postoperative stroke, deep venous thrombosis, pulmonary embolism, myocardial infarction, or intracardiac thrombus. Postoperative dialysis was significantly less likely in patients administered PCC versus rFVIIa following propensity matching (odds ratio = 0.3, 95% CI 0.1-0.7). No significant difference in 30-day mortality in patients receiving PCC versus rFVIIa was present following propensity matching. Use of rFVIIa versus inactive PCCs was significantly associated with renal failure requiring dialysis and increased postoperative bleeding and transfusions. Copyright © 2018 Elsevier Inc. All rights reserved.

  17. Solubilization and folding of a fully active recombinant Gaussia luciferase with native disulfide bonds by using a SEP-Tag.

    Science.gov (United States)

    Rathnayaka, Tharangani; Tawa, Minako; Nakamura, Takashi; Sohya, Shihori; Kuwajima, Kunihiro; Yohda, Masafumi; Kuroda, Yutaka

    2011-12-01

    Gaussia luciferase (GLuc) is the smallest known bioluminescent protein and is attracting much attention as a potential reporter protein. However, its 10 disulfide bond forming cysteines have hampered the efficient production of recombinant GLuc and thus limited its use in bio-imaging application. Here, we demonstrate that the addition of a short solubility enhancement peptide tag (SEP-Tag) to the C-terminus of GLuc (GLuc-C9D) significantly increased the fraction of soluble protein at a standard expression temperature. The expression time was much shorter, and the final yield of GLuc-C9D was significantly higher than with our previous pCold expression system. Reversed phase HPLC indicated that the GLuc-C9D variant folded with a single disulfide bond pattern after proper oxidization. Further, the thermal denaturation of GLuc-C9D was completely reversible, and its secondary structure content remained unchanged until 40°C as assessed by CD spectroscopy. The (1)H-NMR spectrum of GLuc indicated sharp well dispersed peaks typical for natively folded proteins. GLuc-C9D bioluminescence activity was strong and fully retained even after incubation at high temperatures. These results suggest that solubilization using SEP-Tags can be useful for producing large quantities of proteins containing multiple disulfide bonds. Copyright © 2011. Published by Elsevier B.V.

  18. Efficacy and safety of a modified intravenous recombinant tissue plasminogen activator regimen in Chinese patients with acute ischemic stroke.

    Science.gov (United States)

    Pan, Shu-Ming; Liu, Jia-Fu; Liu, Ming; Shen, Sa; Li, Hao-Jun; Dai, Li-Hua; Chen, Xiang-Jun

    2013-07-01

    Thrombolytic treatment with intravenous (IV) recombinant tissue plasminogen activator (rtPA; 0.90 mg/kg, with a maximum dose of 90 mg) has been recommended as the standard management for acute ischemic stroke (AIS) thrombolysis. However, the dose of IV rtPA in Asia remains controversial. This study was designed to verify the safety and efficacy of IV rtPA treatment for AIS with a lower dosage (0.90 mg/kg, with a maximum dose of 50 mg). Patients were divided into 3 dosage groups according to body weight (BW): group 1, 67 kg for descent were included in the study. The baseline characteristics of the 3 dosage groups were well matched. In group 1 (BW 67 kg for <0.75 mg/kg; n = 31; P = .362). There were no significantly statistical differences in the incidence of symptomatic intracerebral hemorrhage and mortality rate. This IV rtPA regimen (0.90 mg/kg, with a maximum dose of 50 mg) not only shows sufficient favorable outcome in clinical practice in Chinese patients with AIS but also good health economic savings. This regimen could be suitable for many developing countries. Copyright © 2013 National Stroke Association. Published by Elsevier Inc. All rights reserved.

  19. Effects of edaravone on early outcomes in acute ischemic stroke patients treated with recombinant tissue plasminogen activator.

    Science.gov (United States)

    Wada, Tomoki; Yasunaga, Hideo; Inokuchi, Ryota; Horiguchi, Hiromasa; Fushimi, Kiyohide; Matsubara, Takehiro; Nakajima, Susumu; Yahagi, Naoki

    2014-10-15

    We investigated whether edaravone could improve early outcomes in acute ischemic stroke patients treated with recombinant tissue plasminogen activator (rtPA). We conducted a retrospective cohort study using the Japanese Diagnosis Procedure Combination database. We identified patients admitted with a primary diagnosis of ischemic stroke from 1 July 2010 to 31 March 2012 and treated with rtPA on the same day of stroke onset or the following day. Thereafter, we selected those who received edaravone on the same day of rtPA administration (edaravone group), and those who received rtPA without edaravone (control group). The primary outcomes were modified Rankin Scale (mRS) scores at discharge. One-to-one propensity-score matching was performed between the edaravone and control groups. An ordinal logistic regression analysis for mRS scores at discharge was performed with adjustment for possible variables as well as clustering of patients within hospitals using a generalized estimating equation. We identified 6336 eligible patients for inclusion in the edaravone group (n=5979; 94%) and the control group (n=357; 6%) as the total population. In 356 pairs of the propensity-matched population, the ordinal logistic regression analysis showed that edaravone was significantly associated with lower mRS scores of patients at discharge (adjusted odds ratio: 0.74; 95% confidence interval: 0.57-0.96). Edaravone may improve early outcomes in acute ischemic stroke patients treated with rtPA. Copyright © 2014 Elsevier B.V. All rights reserved.

  20. Expression, purification, and refolding of active recombinant human E-selectin lectin and EGF domains in Escherichia coli.

    Science.gov (United States)

    Kawano, Susumu; Iyaguchi, Daisuke; Okada, Chiaki; Sasaki, Yusuke; Toyota, Eiko

    2013-06-01

    Attempts to obtain active E-selectin from Escherichia coli (E. coli) have not yet been successful. In this study, we succeeded in expressing the recombinant lectin and epidermal growth factor domain fragments of human E-selectin (rh-ESLE) in E. coli on a large-scale. The rh-ESLE protein was expressed as an inactive form in the inclusion bodies. The inactive form of rh-ESLE was denatured and solubilized by 6 M guanidine hydrochloride and then purified by Ni(2+) affinity chromatography under denaturing conditions. Denatured rh-ESLE was then refolded by a rapid-dilution method using a large amount of refolding buffer, which contained arginine and cysteine/cystine. The refolded rh-ESLE showed binding affinity for sLe(X) (K(d) = 321 nM, B(max) = 1.9 pmol/μg protein). This result suggests that the refolded rh-ESLE recovered its native and functional structure.

  1. Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs

    Science.gov (United States)

    Dumont, Jennifer A.; Liu, Tongyao; Low, Susan C.; Zhang, Xin; Kamphaus, George; Sakorafas, Paul; Fraley, Cara; Drager, Douglas; Reidy, Thomas; McCue, Justin; Franck, Helen W. G.; Merricks, Elizabeth P.; Nichols, Timothy C.; Bitonti, Alan J.; Pierce, Glenn F.

    2012-01-01

    Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation. PMID:22246033

  2. Neutrophils Release Metalloproteinases during Adhesion in the Presence of Insulin, but Cathepsin G in the Presence of Glucagon

    Directory of Open Access Journals (Sweden)

    Natalia V. Fedorova

    2018-01-01

    Full Text Available In patients with reperfusion after ischemia and early development of diabetes, neutrophils can attach to blood vessel walls and release their aggressive bactericide agents, which damage the vascular walls. Insulin and 17β-estradiol (E2 relieve the vascular complications observed in metabolic disorders. In contrast, glucagon plays an essential role in the pathophysiology of diabetes. We studied the effect of hormones on neutrophil secretion during adhesion to fibronectin. Amino acid analysis revealed that proteins secreted by neutrophils are characterized by a stable amino acid profile enriched with glutamate, leucine, lysine, and arginine. The total amount of secreted proteins defined as the sum of detected amino acids was increased in the presence of insulin and reduced in the presence of glucagon. E2 did not affect the amount of protein secretion. Proteome analysis showed that in the presence of insulin and E2, neutrophils secreted metalloproteinases MMP-9 and MMP-8 playing a key role in modulation of the extracellular matrix. In contrast, glucagon induced the secretion of cathepsin G, a key bactericide protease of neutrophils. Cathepsin G can promote the development of vascular complications because of its proinflammatory activity and ability to stimulate neutrophil adhesion via the proteolysis of surface receptors.

  3. A pro-cathepsin L mutant is a luminal substrate for endoplasmic-reticulum-associated degradation in C. elegans.

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    Mark T Miedel

    Full Text Available Endoplasmic-reticulum associated degradation (ERAD is a major cellular misfolded protein disposal pathway that is well conserved from yeast to mammals. In yeast, a mutant of carboxypeptidase Y (CPY* was found to be a luminal ER substrate and has served as a useful marker to help identify modifiers of the ERAD pathway. Due to its ease of genetic manipulation and the ability to conduct a genome wide screen for modifiers of molecular pathways, C. elegans has become one of the preferred metazoans for studying cell biological processes, such as ERAD. However, a marker of ERAD activity comparable to CPY* has not been developed for this model system. We describe a mutant of pro-cathepsin L fused to YFP that no longer targets to the lysosome, but is efficiently eliminated by the ERAD pathway. Using this mutant pro-cathepsin L, we found that components of the mammalian ERAD system that participate in the degradation of ER luminal substrates were conserved in C. elegans. This transgenic line will facilitate high-throughput genetic or pharmacological screens for ERAD modifiers using widefield epifluorescence microscopy.

  4. Rheumatoid Factor Positivity Is Associated with Increased Joint Destruction and Upregulation of Matrix Metalloproteinase 9 and Cathepsin K Gene Expression in the Peripheral Blood in Rheumatoid Arthritic Patients Treated with Methotrexate

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2013-01-01

    Full Text Available We evaluated changes in gene expression of mTOR, p21, caspase-3, ULK1, TNFα, matrix metalloproteinase (MMP-9, and cathepsin K in the whole blood of rheumatoid arthritic (RA patients treated with methotrexate (MTX in relation to their rheumatoid factor status, clinical, immunological, and radiological parameters, and therapeutic response after a 24-month follow-up. The study group consisted of 35 control subjects and 33 RA patients without previous history of MTX treatment. Gene expression was measured using real-time RT-PCR. Decreased disease activity in patients at the end of the study was associated with significant downregulation of TNFα expression. Downregulation of mTOR was observed in seronegative patients, while no significant changes in the expression of p21, ULK1, or caspase-3 were noted in any RA patients at the end of the study. The increase in erosion numbers observed in the seropositive patients at the end of the follow-up was accompanied by upregulation of MMP-9 and cathepsin K, while seronegative patients demonstrated an absence of significant changes in MMP-9 and cathepsin K expression and no increase in the erosion score. Our results suggest that increased expression of MMP-9 and cathepsin K genes in the peripheral blood might indicate higher bone tissue destruction activity in RA patients treated with methotrexate. The clinical study registration number is 0120.0810610.

  5. Newly diagnosed congenital factor VII deficiency and utilization of recombinant activated factor VII (NovoSeven(®)).

    Science.gov (United States)

    Bartosh, Nicole S; Tomlin, Tara; Cable, Christian; Halka, Kathleen

    2013-01-01

    This case report presents a newly diagnosed congenital factor VII deficiency treated with recombinant activated factor VII (rFVIIa). Congenital factor VII deficiency is a rare autosomal-recessive bleeding disorder that occurs in fewer than 1/500,000 persons. Its presentation can vary from epistaxis to hemarthroses and severe central nervous system bleeding, and correlates poorly with factor VII levels. Our patient had not had a significant hemostatic challenge prior to his presentation and therefore never had any symptomatology suggestive of this disease. He was treated with rFVIIa, and was able to undergo repair of his fractures without bleeding. A 19-year-old African-American male presented to the emergency room after an altercation that resulted in significant trauma. He sustained bilateral mandibular angle fractures and orbital floor fractures, requiring urgent surgical correction. On initial evaluation, he was noted to have a prolonged prothrombin time of 40.1 seconds, with an International Normalized Ratio of 4.0, a normal activated partial thromboplastin time of 29.9 seconds, and a platelet count of 241. After receiving vitamin K and fresh frozen plasma, he was taken to the operating room for a temporary rigid maxillomandibular fixation. A 1:1 mixing study with normal plasma corrected the prothrombin time (decreasing from 40.7 to 14.7 seconds) and a factor VII assay revealed 5% of the normal factor VII level. The patient was diagnosed with congenital factor VII deficiency. Due to his coagulopathy and the extensive surgical correction needed, rFVIIa was administered and surgery was accomplished without hemorrhagic sequelae. This case report and review describes a rare congenital disease, the history of rFVIIa use, and its mechanism. rFVIIA use in our patient provided a treatment option that allowed the necessary surgical correction, but further prospective studies on dose optimization would ensure adequate dosing with minimal risk of severe side effects.

  6. Newly diagnosed congenital factor VII deficiency and utilization of recombinant activated factor VII (NovoSeven®)

    Science.gov (United States)

    Bartosh, Nicole S; Tomlin, Tara; Cable, Christian; Halka, Kathleen

    2013-01-01

    This case report presents a newly diagnosed congenital factor VII deficiency treated with recombinant activated factor VII (rFVIIa). Congenital factor VII deficiency is a rare autosomal-recessive bleeding disorder that occurs in fewer than 1/500,000 persons. Its presentation can vary from epistaxis to hemarthroses and severe central nervous system bleeding, and correlates poorly with factor VII levels. Our patient had not had a significant hemostatic challenge prior to his presentation and therefore never had any symptomatology suggestive of this disease. He was treated with rFVIIa, and was able to undergo repair of his fractures without bleeding. Case report A 19-year-old African-American male presented to the emergency room after an altercation that resulted in significant trauma. He sustained bilateral mandibular angle fractures and orbital floor fractures, requiring urgent surgical correction. On initial evaluation, he was noted to have a prolonged prothrombin time of 40.1 seconds, with an International Normalized Ratio of 4.0, a normal activated partial thromboplastin time of 29.9 seconds, and a platelet count of 241. After receiving vitamin K and fresh frozen plasma, he was taken to the operating room for a temporary rigid maxillomandibular fixation. A 1:1 mixing study with normal plasma corrected the prothrombin time (decreasing from 40.7 to 14.7 seconds) and a factor VII assay revealed 5% of the normal factor VII level. The patient was diagnosed with congenital factor VII deficiency. Due to his coagulopathy and the extensive surgical correction needed, rFVIIa was administered and surgery was accomplished without hemorrhagic sequelae. Conclusion This case report and review describes a rare congenital disease, the history of rFVIIa use, and its mechanism. rFVIIA use in our patient provided a treatment option that allowed the necessary surgical correction, but further prospective studies on dose optimization would ensure adequate dosing with minimal risk of

  7. The next generation of sepsis clinical trial designs: what is next after the demise of recombinant human activated protein C?*.

    Science.gov (United States)

    Opal, Steven M; Dellinger, R Phillip; Vincent, Jean-Louis; Masur, Henry; Angus, Derek C

    2014-07-01

    The developmental pipeline for novel therapeutics to treat sepsis has diminished to a trickle compared to previous years of sepsis research. While enormous strides have been made in understanding the basic molecular mechanisms that underlie the pathophysiology of sepsis, a long list of novel agents have now been tested in clinical trials without a single immunomodulating therapy showing consistent benefit. The only antisepsis agent to successfully complete a phase III clinical trial was human recumbent activated protein C. This drug was taken off the market after a follow-up placebo-controlled trial (human recombinant activated Protein C Worldwide Evaluation of Severe Sepsis and septic Shock [PROWESS SHOCK]) failed to replicate the favorable results of the initial registration trial performed ten years earlier. We must critically reevaluate our basic approach to the preclinical and clinical evaluation of new sepsis therapies. We selected the major clinical studies that investigated interventional trials with novel therapies to treat sepsis over the last 30 years. Phase II and phase III trials investigating new treatments for sepsis and editorials and critiques of these studies. Selected manuscripts and clinical study reports were analyzed from sepsis trials. Specific shortcomings and potential pit falls in preclinical evaluation and clinical study design and analysis were reviewed and synthesized. After review and discussion, a series of 12 recommendations were generated with suggestions to guide future studies with new treatments for sepsis. We need to improve our ability to define appropriate molecular targets for preclinical development and develop better methods to determine the clinical value of novel sepsis agents. Clinical trials must have realistic sample sizes and meaningful endpoints. Biomarker-driven studies should be considered to categorize specific "at risk" populations most likely to benefit from a new treatment. Innovations in clinical trial design

  8. Efficacy and effectiveness of recombinant human activated protein C in severe sepsis of adults

    Directory of Open Access Journals (Sweden)

    Greiner, Wolfgang

    2007-07-01

    Full Text Available Introduction: Sepsis is defined as an invasion of microorganisms and/or their toxins into the blood associated the reaction of the organism to this invasion. Severe sepsis is a major cost driver in intensive care medicine. In Germany, prevalence data was assessed in the context of the German Prevalence Study. Severe sepsis has a prevalence of 35% in German intensive care units. Research questions: The following questions were analysed: is Drotrecogin alfa (activated (DAA effective in the treatment of patients with severe sepsis and a mixed risk of death, both in all patients and in different subgroups? Is DAA effective in the treatment of patients with severe sepsis and low risk of death? Is DAA cost effective in the treatment of patients with severe sepsis compared to placebo? Methods: Only studies with adult patients are included. There are no other exclusion criteria. A systematic literature search is performed by the German Institute of Medical Documentation and Information (DIMDI. The literature search yielded as a total of 847 hits. After screening of the abstracts, 165 medical and 101 economic publications were chosen for full text appraisal. Results: Therapy with DAA appears to be cost effective in reducing 28-day-mortality in patients with severe sepsis and a high risk of death. A high risk of death is indicated by the presence of multiorgan failure (≥2 and/or an APACHE-II-Score ≥25. Therapy with DAA is not associated with a long-term reduction of mortality at later follow-up assessments. Therapy with DAA is not associated with a long-term reduction of mortality at later follow-up assessments. Therapy with DAA is cost-effective in patients with multiorgan failure and/or an APACHE II Score (≥25. In patients with a lower risk of death, DAA is not cost-effective. Costs associated with bleeding events have been rarely included in cost calculations. Discussion: DAA appears to reduce mortality in patients with severe sepsis and a high

  9. Recombinational repair: workshop summary

    International Nuclear Information System (INIS)

    Howard-Flanders, P.

    1983-01-01

    Recombinational repair may or may not be synonymous with postreplication repair. Considerable progress has been made in the study of the relevant enzymes, particularly those from bacteria. In this workshop we focus on the recombination enzyme RecA protein. What structural changes take place in the protein and in DNA during repair. How does homologous pairing take place. How is ATP hydrolysis coupled to the stand exchange reaction and the formation of heteroduplx DNA. Turning to another enzyme needed for certain kinds of bacterial recombination, we will ask whether the purified recB protein and recC protein complement each other and are sufficient for exonuclease V activity. In higher cells, we would like to know whether sister exchanges, which occur in bacteria after uv irradiation, are also seen in animal cells

  10. [HPV DNA vaccines expressing recombinant CRT/HPV6bE7 fusion protein inhibit tumor growth and angiogenic activity].

    Science.gov (United States)

    Xu, Yan; Cheng, Hao; Zhao, Ke-Jia; Zhu, Ke-Jian; Zhang, Xing

    2007-11-01

    This paper was to study the angiogenic inhibitory effect and the potential antitumor effect of the constructed recombinant DNA vaccine CRT/HPV6bE7 in vivo. The C57BL/6 mice were vaccinated respectively with recombinant CRT/HPV6bE7 DNA plamids. The inhibitory effects on angiogenesis of generated vaccines in vivo were evaluated by a bFGF-induced angiogenesis assay using the Matrigel kit. To investigate the potential antitumor effect, the mean tumor weights, sizes and tumor appearing times were measured in C57BL/6 mice treated with HPV6bE7-expressing B16 cells. The results indicated that the recombinants CRT180/HPV6bE7 and CRT180 showed strong anti-angiogenic effects in bFGF-induced angiogenesis in vivo. Moreover, CRT180/HPV6bE7 and CRT180 DNA vaccines could significantly inhibit the tumor growth in tumor challenge experiment, and CRT180/HPV6bE7 was superior to other vaccines in delaying tumor formation time, limiting tumor size and weight in tumor protection experiment. In conclusion, recombinant CRT180/HPV6bE7 DNA could elicit a most efficient anti-angiogenic effect and inhibit tumor growth in mice inoculated with DNA vaccines. The antiangiogenic activity of CRT were suggested residing in a domain between CRT 120-180 aa.

  11. Origin of the CMS gene locus in rapeseed cybrid mitochondria: active and inactive recombination produces the complex CMS gene region in the mitochondrial genomes of Brassicaceae.

    Science.gov (United States)

    Oshima, Masao; Kikuchi, Rie; Imamura, Jun; Handa, Hirokazu

    2010-01-01

    CMS (cytoplasmic male sterile) rapeseed is produced by asymmetrical somatic cell fusion between the Brassica napus cv. Westar and the Raphanus sativus Kosena CMS line (Kosena radish). The CMS rapeseed contains a CMS gene, orf125, which is derived from Kosena radish. Our sequence analyses revealed that the orf125 region in CMS rapeseed originated from recombination between the orf125/orfB region and the nad1C/ccmFN1 region by way of a 63 bp repeat. A precise sequence comparison among the related sequences in CMS rapeseed, Kosena radish and normal rapeseed showed that the orf125 region in CMS rapeseed consisted of the Kosena orf125/orfB region and the rapeseed nad1C/ccmFN1 region, even though Kosena radish had both the orf125/orfB region and the nad1C/ccmFN1 region in its mitochondrial genome. We also identified three tandem repeat sequences in the regions surrounding orf125, including a 63 bp repeat, which were involved in several recombination events. Interestingly, differences in the recombination activity for each repeat sequence were observed, even though these sequences were located adjacent to each other in the mitochondrial genome. We report results indicating that recombination events within the mitochondrial genomes are regulated at the level of specific repeat sequences depending on the cellular environment.

  12. Production of Active Nonglycosylated Recombinant B-Chain of Type-2 Ribosome-Inactivating Protein from Viscum articulatum and Its Biological Effects on Peripheral Blood Mononuclear Cells

    Directory of Open Access Journals (Sweden)

    Tzu-Li Lu

    2011-01-01

    Full Text Available Type-2 ribosome-inactivating proteins, composed of a toxic A-chain and lectin-like B-chain, display various biological functions, including cytotoxicity and immunomodulation. We here cloned the lectin-like B-chain encoding fragment of a newly identified type-2 RIP gene, articulatin gene, from Viscum articulatum, into a bacterial expression vector to obtain nonglycosylated recombinant protein expressed in inclusion bodies. After purification and protein refolding, soluble refolded recombinant articulatin B-chain (rATB showed lectin activity specific toward galactoside moiety and was stably maintained while stored in low ionic strength solution. Despite lacking glycosylation, rATB actively bound leukocytes with preferential binding to monocytes and in vitro stimulated PBMCs to release cytokines without obvious cytotoxicity. These results implicated such a B-chain fragment as a potential immunomodulator.

  13. Production and characterization of monoclonal antibodies against cathepsin B and cathepsin B-Like proteins of Naegleria fowleri.

    Science.gov (United States)

    Seong, Gi-Sang; Sohn, Hae-Jin; Kang, Heekyoung; Seo, Ga-Eun; Kim, Jong-Hyun; Shin, Ho-Joon

    2017-12-01

    Naegleria fowleri causes fatal primary amoebic meningoencephalitis (PAM) in humans and experimental animals. In previous studies, cathepsin B (nfcpb) and cathepsin B-like (nfcpb-L) genes of N. fowleri were cloned, and it was suggested that refolding rNfCPB and rNfCPB-L proteins could play important roles in host tissue invasion, immune response evasion and nutrient uptake. In this study, we produced anti-NfCPB and anti-NfCPB-L monoclonal antibodies (McAb) using a cell fusion technique, and observed their immunological characteristics. Seven hybridoma cells secreting rNfCPB McAbs and three hybridoma cells secreting rNfCPB-L McAbs were produced. Among these, 2C9 (monoclone for rNfCPB) and 1C8 (monoclone for rNfCPB-L) McAb showed high antibody titres and were finally selected for use. As determined by western blotting, 2C9 McAb bound to N. fowleri lysates, specifically the rNfCPB protein, which had bands of 28 kDa and 38.4 kDa. 1C8 McAb reacted with N. fowleri lysates, specifically the rNfCPB-L protein, which had bands of 24 kDa and 34 kDa. 2C9 and 1C8 monoclonal antibodies did not bind to lysates of other amoebae, such as N. gruberi, Acanthamoeba castellanii and A. polyphaga in western blot analyses. Immuno-cytochemistry analysis detected NfCPB and NfCPB-L proteins in the cytoplasm of N. fowleri trophozoites, particularly in the pseudopodia and food-cup. These results suggest that monoclonal antibodies produced against rNfCPB and rNfCPB-L proteins may be useful for further immunological study of PAM. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Administration of recombinant activated factor VII in the intensive care unit after complex cardiovascular surgery: clinical and economic outcomes.

    Science.gov (United States)

    Uber, Walter E; Toole, John M; Stroud, Martha R; Haney, Jason S; Lazarchick, John; Crawford, Fred A; Ikonomidis, John S

    2011-06-01

    Refractory bleeding after complex cardiovascular surgery often leads to increased length of stay, cost, morbidity, and mortality. Recombinant activated factor VII administered in the intensive care unit can reduce bleeding, transfusion, and surgical re-exploration. We retrospectively compared factor VII administration in the intensive care unit with reoperation for refractory bleeding after complex cardiovascular surgery. From 1501 patients who underwent cardiovascular procedures between December 2003 and September 2007, 415 high-risk patients were identified. From this cohort, 24 patients were divided into 2 groups based on whether they either received factor VII in the intensive care unit (n = 12) or underwent reoperation (n = 12) for refractory bleeding. Preoperative and postoperative data were collected to compare efficacy, safety, and economic outcomes. In-hospital survival for both groups was 100%. Factor VII was comparable with reoperation in achieving hemostasis, with both groups demonstrating decreases in chest tube output and need for blood products. Freedom from reoperation was achieved in 75% of patients receiving factor VII, whereas reoperation was effective in achieving hemostasis alone in 83.3% of patients. Prothrombin time, international normalized ratio, and median operating room time were significantly less (P factor VII. Both groups had no statistically significant differences in other efficacy, safety, or economic outcomes. Factor VII administration in the intensive care unit appears comparable with reoperation for refractory bleeding after complex cardiovascular surgical procedures and might represent an alternative to reoperation in selected patients. Future prospective, randomized controlled trials might further define its role. Copyright © 2011 The American Association for Thoracic Surgery. Published by Mosby, Inc. All rights reserved.

  15. 1H NMR structural characterization of a recombinant kringle 2 domain from human tissue-type plasminogen activator

    International Nuclear Information System (INIS)

    Byeon, I.J.L.; Llinas, M.; Kelley, R.F.

    1989-01-01

    The kringle 2 domain of human tissue-type plasminogen activator (t-PA) has been characterized via 1 H NMR spectroscopy at 300 and 620 MHz. The experiments were performed on the isolated domain obtained by expression of the 174-263 portion of t-PA in Escherichia coli. The spectrum of t-Pa kringle 2 is characteristic of a globular structure and shows overall similarity to that of the plasminogen (PGN) kringle 4. Spectral comparison with human and bovine PGN kringle 4 identified side-chain resonances from Leu 46 , which afford a fingerprint of kringle folding, and from most of the aromatic ring spin systems. Ligand-binding studies confirm that t-PA kringle 2 binds L-lysine with an association constant K a ∼ 11.9 mM -1 . The data indicate that homologous or conserved residues relative to those that compose the lysine-binding sites of PGN kringles 1 and 4 are involved in the binding of L-lysine to t-PA kringle 2. These include Tyr 36 and, within the kringle inner loop, Trp 62 , His 64 , Trp 72 , and Tyr 74 . Several labile NH protons of t-PA kringle 2 exhibit retarded H-exchange kinetics, requiring more than a week in 2 H 2 O for full deuteration in the presence of L-lysine at 37 degree C. This reveals that kringle 2 is endowed with a compact, dynamically stable conformation. Proton Overhauser experiments in 1 H 2 O, centered on well-resolved NH resonances between 9.8 and 12 ppm, identify signals arising from the His 48a imidazole NH3 proton and the three Trp indole NH1 protons. Overall, the data indicate a highly structured conformation for the recombinant t-PA kringle 2 that is closely related to that of the previously investigated PGN kringles 1, 4, and 5

  16. Embryonic stem cells deficient for Brca2 or Blm exhibit divergent genotoxic profiles that support opposing activities during homologous recombination

    Energy Technology Data Exchange (ETDEWEB)

    Marple, Teresa [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive San Antonio, TX 78245-3207 (United States); Kim, Tae Moon [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive San Antonio, TX 78245-3207 (United States); Hasty, Paul [Department of Molecular Medicine and Institute of Biotechnology, University of Texas Health Science Center at San Antonio, 15355 Lambda Drive San Antonio, TX 78245-3207 (United States)]. E-mail: hastye@uthscsa.edu

    2006-12-01

    The breast cancer susceptibility protein, Brca2 and the RecQ helicase, Blm (Bloom syndrome mutated) are tumor suppressors that maintain genome integrity, at least in part, through homologous recombination (HR). Brca2 facilitates HR by interacting with Rad51 in multiple regions, the BRC motifs encoded by exon 11 and a single domain encoded by exon 27; however, the exact importance of these regions is not fully understood. Blm also interacts with Rad51 and appears to suppress HR in most circumstances; however, its yeast homologue Sgs1 facilitates HR in response to some genotoxins. To better understand the biological importance of these two proteins, we performed a genotoxic screen on mouse embryonic stem (ES) cells impaired for either Brca2 or Blm to establish their genotoxic profiles (a cellular dose-response to a wide range of agents). This is the first side-by-side comparison of these two proteins in an identical genetic background. We compared cells deleted for Brca2 exon 27 to cells reduced for Blm expression and find that the Brca2- and Blm-impaired cells exhibit genotoxic profiles that reflect opposing activities during HR. Cells deleted for Brca2 exon 27 are hypersensitive to {gamma}-radiation, streptonigrin, mitomycin C and camptothecin and mildly resistant to ICRF-193 which is similar to HR defective cells null for Rad54. By contrast, Blm-impaired cells are hypersensitive to ICRF-193, mildly resistant to camptothecin and mitomycin C and more strongly resistant to hydroxyurea. These divergent profiles support the notion that Brca2 and Blm perform opposing functions during HR in mouse ES cells.

  17. Pharmacokinetics and pharmacodynamics of the cathepsin S inhibitor, LY3000328, in healthy subjects.

    Science.gov (United States)

    Payne, Christopher D; Deeg, Mark A; Chan, Melanie; Tan, Lai Hock; LaBell, Elizabeth Smith; Shen, Tong; DeBrota, David J

    2014-12-01

    The aim of this study was to assess the safety and tolerability, pharmacokinetics and pharmacodynamics of LY3000328 when administered as single escalating doses to healthy volunteers. This was a phase 1, placebo-controlled, dose escalation study with LY3000328 in 21 healthy male volunteers. Subjects were administered escalating LY3000328 doses up to 300 mg with food in this single dose study. Blood samples were collected at set times post-dose for the assessment of LY3000328 pharmacokinetics and the measurement of cathepsin S (CatS) activity, CatS mass and calculated CatS specific activity. All doses of LY3000328 were well tolerated, with linear pharmacokinetics up to the 300 mg dose. The pharmacodynamic activity of LY3000328 was measured ex vivo showing a biphasic response to LY3000328, where CatS activity declines, then returns to baseline, and then increases to a level above baseline. CatS mass was also assessed post-dose which increased in a dose-dependent manner, and continued to increase after LY3000328 had been cleared from the body. CatS specific activity was additionally calculated to normalize CatS activity for changes in CatS mass. This demonstrated the increase in CatS activity was attributable to the increase in CatS mass detected in plasma. A specific inhibitor of CatS which is cleared quickly from plasma may produce a transient decrease in plasma CatS activity which is followed by a more prolonged increase in plasma CatS mass which may have implications for the future clinical development of inhibitors of CatS. © 2014 The British Pharmacological Society.

  18. First-principles study of Frenkel pair recombination in tungsten

    International Nuclear Information System (INIS)

    Qin, Shi-Yao; Jin, Shuo; Li, Yu-Hao; Zhou, Hong-Bo; Zhang, Ying; Lu, Guang-Hong

    2017-01-01

    The recombination of one Frenkel pair in tungsten has been investigated through first-principles simulation. Two different recombination types have been identified: instantaneous and thermally activated. The small recombination barriers for thermally activated recombination cases indicate that recombination can occur easily with a slightly increased temperature. For both of the two recombination types, recombination occurs through the self-interstitial atom moving towards the vacancy. The recombination process can be direct or through replacement sequences, depending on the vertical distance between the vacancy and the 〈1 1 1〉 line of self-interstitial atom pair.

  19. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease.

    Science.gov (United States)

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica.

  20. Immunodiagnosis of Fasciola gigantica Infection Using Monoclonal Antibody-Based Sandwich ELISA and Immunochromatographic Assay for Detection of Circulating Cathepsin L1 Protease

    Science.gov (United States)

    Anuracpreeda, Panat; Chawengkirttikul, Runglawan; Sobhon, Prasert

    2016-01-01

    Background Tropical fasciolosis caused by Fasciola gigantica infection is one of the major diseases infecting ruminants in the tropical regions of Africa and Asia including Thailand. Parasitological diagnosis of fasciolosis is often unreliable and possesses low sensitivity. Therefore, the detection of circulating parasite antigens is thought to be a better alternative for diagnosis of fasciolosis, as it reflects the real parasite burden. Methods In this study, we have produced a monoclonal antibody (MoAb) against recombinant F. gigantica cathepsin L1 (rFgCatL1), and developed both sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunochromatographic (IC) test for rapid detection of circulating cathepsin L1 protease (CatL1) in the sera from mice experimentally and cattle naturally infected with Fasciola gigantica. MoAb 4E3 and biotinylated rabbit anti-recombinant CatL1 antibody were selected due to their high reactivities and specificities. Results The lower detection limits of sandwich ELISA and IC test were 3 pg/ml and 0.256 ng/ml, respectively. Sandwich ELISA and IC test could detect F. gigantica infection from day 1 to 35 post infection. In experimental mice, the sensitivity, specificity and accuracy were 95%, 100% and 98.6% (for sandwich ELISA), and 93%, 100% and 98.2% (for IC test), while in natural cattle they were 98.3%, 100% and 99.5% (for sandwich ELISA), and 96.7%, 100% and 99.1% (for IC test). Conclusions These two assay methods showed high efficiencies and precisions for diagnosis of fasciolosis by F. gigantica. PMID:26731402

  1. Prevalence and clinical significance of cathepsin G antibodies in systemic sclerosis

    Directory of Open Access Journals (Sweden)

    M. Favaro

    2011-09-01

    Full Text Available Objectives: To evaluate the prevalence and clinical significance of cathepsin G antibodies in patients affected with systemic sclerosis (SSc, scleroderma. Methods: 115 patients affected by SSc, 55 (47,8% with diffuse scleroderma (dSSc and 60 (52,2% with limited scleroderma (lSSc, were tested for cathepsin G antibodies by ELISA method. Moreover these sera were evaluated by indirect immunofluorescence (IIF on ethanol and formalin fixed human neutrophils. Results: By means of the ELISA method 16 (13,9% patients were found to be sera positive for anti-cathepsin G, 2 (12.5% of which showed a perinuclear fluorescence pattern (P-ANCA and 4 (25% an atypical ANCA staining, while 10 (62,5% were negative on IIF. The IIF on scleroderma sera revealed 5 (4,3% P-ANCA and 18 (15,7% atypical ANCA patterns. The anti-cathepsin G antibodies significantly prevailed in scleroderma sera (p=0.02 when their frequency was compared with that of healthy controls; while they were not significantly associated to any clinical or serological features of SSc patients. Conclusions: The anti-cathepsin G antibodies were significantly frequent in scleroderma sera; however, no clinical correlations were found. Thus, the significance of their presence in SSc still needs to be clarified.

  2. Identification, immunolocalization, and characterization analyses of an exopeptidase of papain superfamily, (cathepsin C) from Clonorchis sinensis.

    Science.gov (United States)

    Liang, Pei; He, Lei; Xu, Yanquan; Chen, Xueqing; Huang, Yan; Ren, Mengyu; Liang, Chi; Li, Xuerong; Xu, Jin; Lu, Gang; Yu, Xinbing

    2014-10-01

    Cathepsin C is an important exopeptidase of papain superfamily and plays a number of great important roles during the parasitic life cycle. The amino acid sequence of cathepsin C from Clonorchis sinensis (C. sinensis) showed 54, 53, and 49% identities to that of Schistosoma japonicum, Schistosoma mansoni, and Homo sapiens, respectively. Phylogenetic analysis utilizing the sequences of papain superfamily of C. sinensis demonstrated that cathepsin C and cathepsin Bs came from a common ancestry. Cathepsin C of C. sinensis (Cscathepsin C) was identified as an excretory/secretory product by Western blot analysis. The results of transcriptional level and translational level of Cscathepsin C at metacercaria stage were higher than that at adult worms. Immunolocalization analysis indicated that Cscathepsin C was specifically distributed in the suckers (oral sucker and ventral sucker), eggs, vitellarium, intestines, and testis of adult worms. In the metacercaria, it was mainly detected on the cyst wall and excretory bladder. Combining with the results mentioned above, it implies that Cscathepsin C may be an essential proteolytic enzyme for proteins digestion of hosts, nutrition assimilation, and immune invasion of C. sinensis. Furthermore, it may be a potential diagnostic antigen and drug target against C. sinensis infection.

  3. Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G

    Directory of Open Access Journals (Sweden)

    Andreas Koeberle

    2018-02-01

    Full Text Available Age-related diseases, such as osteoarthritis, Alzheimer’s disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO pathway. Boswellic acids (BAs are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

  4. Triterpene Acids from Frankincense and Semi-Synthetic Derivatives That Inhibit 5-Lipoxygenase and Cathepsin G.

    Science.gov (United States)

    Koeberle, Andreas; Henkel, Arne; Verhoff, Moritz; Tausch, Lars; König, Stefanie; Fischer, Dagmar; Kather, Nicole; Seitz, Stefanie; Paul, Michael; Jauch, Johann; Werz, Oliver

    2018-02-24

    Age-related diseases, such as osteoarthritis, Alzheimer's disease, diabetes, and cardiovascular disease, are often associated with chronic unresolved inflammation. Neutrophils play central roles in this process by releasing tissue-degenerative proteases, such as cathepsin G, as well as pro-inflammatory leukotrienes produced by the 5-lipoxygenase (5-LO) pathway. Boswellic acids (BAs) are pentacyclic triterpene acids contained in the gum resin of the anti-inflammatory remedy frankincense that target cathepsin G and 5-LO in neutrophils, and might thus represent suitable leads for intervention with age-associated diseases that have a chronic inflammatory component. Here, we investigated whether, in addition to BAs, other triterpene acids from frankincense interfere with 5-LO and cathepsin G. We provide a comprehensive analysis of 17 natural tetra- or pentacyclic triterpene acids for suppression of 5-LO product synthesis in human neutrophils. These triterpene acids were also investigated for their direct interference with 5-LO and cathepsin G in cell-free assays. Furthermore, our studies were expanded to 10 semi-synthetic BA derivatives. Our data reveal that besides BAs, several tetra- and pentacyclic triterpene acids are effective or even superior inhibitors of 5-LO product formation in human neutrophils, and in parallel, inhibit cathepsin G. Their beneficial target profile may qualify triterpene acids as anti-inflammatory natural products and pharmacological leads for intervention with diseases related to aging.

  5. Exogenous cathepsin V protein protects human cardiomyocytes HCM from angiotensin Ⅱ-Induced hypertrophy.

    Science.gov (United States)

    Huang, Kun; Gao, Lu; Yang, Ming; Wang, Jiliang; Wang, Zheng; Wang, Lin; Wang, Guobin; Li, Huili

    2017-08-01

    Angiotensin (Ang) Ⅱ-induced cardiac hypertrophy can deteriorate to heart failure, a leading cause of mortality. Endogenous Cathepsin V (CTSV) has been reported to be cardioprotective against hypertrophy. However, little is known about the effect of exogenous CTSV on cardiac hypertrophy. We used the human cardiomyocytes HCM as a cell model to investigate the effects of exogenous CTSV on Ang Ⅱ-induced cardiac cell hypertrophy. Cell surface area and expression of classical markers of hypertrophy were analyzed. We further explored the mechanism of CTSV cardioprotective by assessing the levels and activities of PI3K/Akt/mTOR and MAPK signaling pathway proteins. We found that pre-treating cardiomyocytes with CTSV could significantly inhibit Ang Ⅱ-induced hypertrophy. The mRNA expression of hypertrophy markers ANP, BNP and β-MHC was obviously elevated in Ang Ⅱ-treated cardiac cells. Whereas, exogenous CTSV effectively halted this elevation. Further study revealed that the protective effects of exogenous CTSV might be mediated by repressing the phosphorylation of proteins in the PI3K/Akt/mTOR and MAPK pathways. Based on our results, we concluded that exogenous CTSV inhibited Ang Ⅱ-induced hypertrophy in HCM cells by inhibiting PI3K/Akt/mTOR. This study provides experimental evidence for the application of CTSV protein for the treatment of cardiac hypertrophy. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Cathepsin L is required for endothelial progenitor cell-induced neovascularization

    Energy Technology Data Exchange (ETDEWEB)

    Urbich, Carmen; Heeschen, Christopher; Aicher, Alexandra; Sasaki, Ken-ichiro; Bruhl, Thomas; Hofmann, Wolf K.; Peters, Christoph; Reinheckel, Thomas; Pennacchio, Len A.; Abolmaali, Nasreddin D.; Chavakis, Emmanouil; Zeiher, Andreas M.; Dimmeler, Stefanie

    2004-01-15

    Infusion of endothelial progenitor cells (EPCs), but not of mature endothelial cells (ECs), promotes neovascularization after ischemia. We performed a gene expression profiling of EPCs and ECs to identify genes, which might be important for the neovascularization capacity of EPCs. Intriguingly, the protease cathepsin L (CathL) was highly expressed in EPCs as opposed to ECs and is essential for matrix degradation and invasion by EPCs in vitro. CathL deficient mice showed impaired functional recovery after hind limb ischemia supporting the concept for an important role of CathL in postnatal neovascularization. Infused CathL deficient progenitor cells failed to home to sites of ischemia and to augment neovascularization. In contrast, over expression of CathL in mature ECs significantly enhanced their invasive activity and induced their neovascularization capacity in vivo. Taken together, CathL plays a crucial role for the integration of circulating EPCs into the ischemic tissue and is required for neovascularization mediated by EPCs.

  7. Alpha-synuclein induces lysosomal rupture and cathepsin dependent reactive oxygen species following endocytosis.

    Directory of Open Access Journals (Sweden)

    David Freeman

    Full Text Available α-synuclein dysregulation is a critical aspect of Parkinson's disease pathology. Recent studies have observed that α-synuclein aggregates are cytotoxic to cells in culture and that this toxicity can be spread between cells. However, the molecular mechanisms governing this cytotoxicity and spread are poorly characterized. Recent studies of viruses and bacteria, which achieve their cytoplasmic entry by rupturing intracellular vesicles, have utilized the redistribution of galectin proteins as a tool to measure vesicle rupture by these organisms. Using this approach, we demonstrate that α-synuclein aggregates can induce the rupture of lysosomes following their endocytosis in neuronal cell lines. This rupture can be induced by the addition of α-synuclein aggregates directly into cells as well as by cell-to-cell transfer of α-synuclein. We also observe that lysosomal rupture by α-synuclein induces a cathepsin B dependent increase in reactive oxygen species (ROS in target cells. Finally, we observe that α-synuclein aggregates can induce inflammasome activation in THP-1 cells. Lysosomal rupture is known to induce mitochondrial dysfunction and inflammation, both of which are well established aspects of Parkinson's disease, thus connecting these aspects of Parkinson's disease to the propagation of α-synuclein pathology in cells.

  8. Cathepsin X in serum from patients with colorectal cancer: relation to prognosis

    Science.gov (United States)

    Vizin, Tjasa; Christensen, Ib Jarle; Nielsen, Hans Jørgen; Kos, Janko

    2012-01-01

    Background Up-regulation of lysosomal cysteine protease cathepsin X (Cat X) is associated with disorders of the immune system and neurodegenerative diseases, while its role in the development and progression of cancer is less understood. Enhanced secretion of pro-Cat X was observed in malignant processes, and therefore, the level of total serum Cat X rather than the active enzyme may better reflect the tumour status. Patients and methods Seventy-seven patients with colorectal cancer (CRC) were included in a retrospective study. Blood samples were collected prior to therapy. Using ELISA, the values of total Cat X were measured in serum. Groups of healthy persons (n=77), patients with adenomas (n=77) and patients with non-neoplastic findings (n=77) were included. Results Significant differences between the group of colorectal patients and the groups of healthy persons, adenoma patients and patients with non-malignant findings could not be shown (p=0.89). Within the group of CRC, higher levels of total Cat X significantly correlated to shorter overall survival (HR=2.08, 95% CI:1.07–4.05, p=0.028). Conclusions Total serum Cat X could be a useful prognostic indicator for determining survival of patients with CRC. Increased serum levels of total Cat X may reflect more aggressive tumour cell phenotypes and suggest the involvement of Cat X in processes involved in later stages of tumour progression. PMID:23077459

  9. High expression of AID and active class switch recombination might account for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease.

    Science.gov (United States)

    Palacios, Florencia; Moreno, Pilar; Morande, Pablo; Abreu, Cecilia; Correa, Agustín; Porro, Valentina; Landoni, Ana Ines; Gabus, Raul; Giordano, Mirta; Dighiero, Guillermo; Pritsch, Otto; Oppezzo, Pablo

    2010-06-03

    Interaction of chronic lymphocytic leukemia (CLL) B cells with tissue microenvironment has been suggested to favor disease progression by promoting malignant B-cell growth. Previous work has shown expression in peripheral blood (PB) of CLL B cells of activation-induced cytidine deaminase (AID) among CLL patients with an unmutated (UM) profile of immunoglobulin genes and with ongoing class switch recombination (CSR) process. Because AID expression results from interaction with activated tissue microenvironment, we speculated whether the small subset with ongoing CSR is responsible for high levels of AID expression and could be derived from this particular microenvironment. In this work, we quantified AID expression and ongoing CSR in PB of 50 CLL patients and characterized the expression of different molecules related to microenvironment interaction. Our results show that among UM patients (1) high AID expression is restricted to the subpopulation of tumoral cells ongoing CSR; (2) this small subset expresses high levels of proliferation, antiapoptotic and progression markers (Ki-67, c-myc, Bcl-2, CD49d, and CCL3/4 chemokines). Overall, this work outlines the importance of a cellular subset in PB of UM CLL patients with a poor clinical outcome, high AID levels, and ongoing CSR, whose presence might be a hallmark of a recent contact with the microenvironment.

  10. Role of teh Rad52 Amino-terminal DNA Binding Activity in DNA Strand Capture in Homologous Recombination

    DEFF Research Database (Denmark)

    Shi, Idina; Hallwyl, Swee Chuang Lim; Seong, Changhyun

    2009-01-01

    Saccharomyces cerevisiae Rad52 protein promotes homologous recombination by nucleating the Rad51 recombinase onto replication protein A-coated single-stranded DNA strands and also by directly annealing such strands. We show that the purified rad52-R70A mutant protein, with a compromised amino-ter...

  11. Inhibitory Effects of Juices Prepared from Individual Vegetables on CYP3A4 Activity in Recombinant CYP3A4 and LS180 Cells.

    Science.gov (United States)

    Tsujimoto, Masayuki; Agawa, Chie; Ueda, Shinya; Yamane, Takayoshi; Kitayama, Haruna; Terao, Aya; Fukuda, Tomoya; Minegaki, Tetsuya; Nishiguchi, Kohshi

    2017-01-01

    Human intestinal absorption and drug metabolism vary to a large extent among individuals. For example, CYP3A4 activity has large individual variation that cannot be attributed to only genetic differences. Various flavonoids in vegetables, such as kaempferol and quercetin, possess inhibitory effects, and some vegetable and fruit juices have also been found to inhibit CYP3A4 activity. Therefore, differences in daily intake of flavonoid-containing vegetables may induce individual variation in intestinal bioavailability. To identify a vegetable that strongly inhibits CYP3A4, we investigated the effects of juices, prepared from individual vegetables, on CYP3A4 activity using recombinant CYP3A4 and LS180 cells in this study. Nine vegetable juices (cabbage, Japanese radish, onion, tomato, eggplant, carrot, Chinese cabbage, green pepper, and lettuce), were prepared and recombinant CYP3A4 and LS180 cells were used for evaluation of CYP3A4 activity. Metabolism to 6β-hydroxytestosterone by recombinant CYP3A4 was strongly inhibited by cabbage, onion, and green pepper juices, and cabbage and green pepper juices significantly inhibited CYP3A4 activity in a preincubation time-dependent manner. In addition, CYP3A4 activity in LS180 cells was significantly inhibited by cabbage and onion juices. In conclusion, this study showed that juices prepared from some individual vegetables could significantly inhibit CYP3A4 activity. Therefore, variation in the daily intake of vegetables such as cabbage and onion may be one of the factors responsible for individual differences in intestinal bioavailability.

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

    Directory of Open Access Journals (Sweden)

    Anna Prudova

    2016-08-01

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

  13. Mannose 6-phosphate-independent targeting of cathepsin D to lysosomes in HepG2 cells

    NARCIS (Netherlands)

    Rijnboutt, S.; Kal, A. J.; Geuze, H. J.; Aerts, H.; Strous, G. J.

    1991-01-01

    We have studied the role of N-linked oligosaccharides and proteolytic processing on the targeting of cathepsin D to the lysosomes in the human hepatoma cell line HepG2. In the presence of tunicamycin cathepsin D was synthesized as an unglycosylated 43-kDa proenzyme which was proteolytically

  14. Managing the SOS Response for Enhanced CRISPR-Cas-Based Recombineering in E. coli through Transient Inhibition of Host RecA Activity.

    Science.gov (United States)

    Moreb, Eirik Adim; Hoover, Benjamin; Yaseen, Adam; Valyasevi, Nisakorn; Roecker, Zoe; Menacho-Melgar, Romel; Lynch, Michael D

    2017-12-15

    Phage-derived "recombineering" methods are utilized for bacterial genome editing. Recombineering results in a heterogeneous population of modified and unmodified chromosomes, and therefore selection methods, such as CRISPR-Cas9, are required to select for edited clones. Cells can evade CRISPR-Cas-induced cell death through recA-mediated induction of the SOS response. The SOS response increases RecA dependent repair as well as mutation rates through induction of the umuDC error prone polymerase. As a result, CRISPR-Cas selection is more efficient in recA mutants. We report an approach to inhibiting the SOS response and RecA activity through the expression of a mutant dominant negative form of RecA, which incorporates into wild type RecA filaments and inhibits activity. Using a plasmid-based system in which Cas9 and recA mutants are coexpressed, we can achieve increased efficiency and consistency of CRISPR-Cas9-mediated selection and recombineering in E. coli, while reducing the induction of the SOS response. To date, this approach has been shown to be independent of recA genotype and host strain lineage. Using this system, we demonstrate increased CRISPR-Cas selection efficacy with over 10 000 guides covering the E. coli chromosome. The use of dominant negative RecA or homologues may be of broad use in bacterial CRISPR-Cas-based genome editing where the SOS pathways are present.

  15. Identification of a recombinant inulin fructotransferase (difructose dianhydride III forming) from Arthrobacter sp. 161MFSha2.1 with high specific activity and remarkable thermostability.

    Science.gov (United States)

    Wang, Xiao; Yu, Shuhuai; Zhang, Tao; Jiang, Bo; Mu, Wanmeng

    2015-04-08

    Difructose dianhydride III (DFA III) is a functional carbohydrate produced from inulin by inulin fructotransferase (IFTase, EC 4.2.2.18). In this work, an IFTase gene from Arthrobacter sp. 161MFSha2.1 was cloned and expressed in Escherachia coli. The recombinant enzyme was purified by metal affinity chromatography. It showed significant inulin hydrolysis activity, and the produced main product from inulin was determined as DFA III by nuclear magnetic resonance analysis. The molecular mass of the purified protein was calculated to be 43 and 125 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration, respectively, suggesting the native enzyme might be a homotrimer. The recombinant enzyme showed maximal activity as 2391 units/mg at pH 6.5 and 55 °C. It displayed the highest thermostability among previously reported IFTases (DFA III forming) and was stable up to 80 °C for 4 h of incubation. The smallest substrate was determined as nystose. The conversion ratio of inulin to DFA III reached 81% when 100 g/L inulin was catalyzed by 80 nM recombinant enzyme for 20 min at pH 6.5 and 55 °C. All of these data indicated that the IFTase (DFA III forming) from Arthrobacter sp. 161MFSha2.1 had great potential for industrial DFA III production.

  16. Sildenafil Decreases BACE1 and Cathepsin B Levels and Reduces APP Amyloidogenic Processing in the SAMP8 Mouse.

    Science.gov (United States)

    Orejana, Lourdes; Barros-Miñones, Lucía; Jordan, Joaquin; Cedazo-Minguez, Angel; Tordera, Rosa M; Aguirre, Norberto; Puerta, Elena

    2015-06-01

    The senescence-accelerated mouse-prone 8 (SAMP8), used as a model of aging, displays many established pathological features of Alzheimer's disease. Cognitive impairments and increased levels of hyperphosphorylated tau are found in the hippocampus of SAMP8 mice along with an increased β-secretase activity and amyloid-β (Aβ) depositions that increase in number and extent with age. Based on a previous study from our laboratory showing an amelioration of cognitive impairments and tau pathology by sildenafil, in this study we tested whether this drug could also modulate the amyloid precursor protein amyloidogenic processing in this mouse model. Our results show that the protein levels of the β-secretases β-site amyloid precursor protein cleaving enzyme 1 and cathepsin B are higher in the hippocampus of 9-month-old SAMP8 mice than those of age-matched senescence-resistant-1. Sildenafil (7.5mg/kg for 4 weeks) attenuated learning and memory impairments shown by SAMP8 mice in the passive avoidance test. The increased expression of β-site amyloid precursor protein cleaving enzyme 1 was also reduced by sildenafil, an effect paralleled to decreases in the activities of two β-site amyloid precursor protein cleaving enzyme 1 modulators, calpain and cyclin-dependent kinase 5 protein. Interestingly, sildenafil enhanced both Akt and glycogen synthase kinase-3β (ser9) phosphorylation, which could be mediating the reduction in cathepsin B levels found in the hippocampus of sildenafil-treated SAMP8 mice. Sildenafil-induced reduction in β-site amyloid precursor protein cleaving enzyme 1 and cathepsin B expression in SAMP8 mice was associated with a decrease in hippocampal Aβ42 levels which, in turn, could mediate the parallel decline in glial fibrillary acidic protein expression observed in these animals. These findings highlight the therapeutic potential of sildenafil in Alzheimer's disease pathogenesis. © The Author 2014. Published by Oxford University Press on behalf of

  17. Cathepsin K expression is increased in oral lichen planus.

    Science.gov (United States)

    Siponen, Maria; Bitu, Carolina Cavalcante; Al-Samadi, Ahmed; Nieminen, Pentti; Salo, Tuula

    2016-11-01

    Oral lichen planus (OLP) is an idiopathic T-cell-mediated mucosal inflammatory disease. Cathepsin K (Cat K) is one of the lysosomal cysteine proteases. It is involved in many pathological conditions, including osteoporosis and cancer. The expression and role of Cat K in OLP are unknown. Twenty-five oral mucosal specimens diagnosed histopathologically as OLP and fourteen healthy controls (HC) were used to study the immunohistochemical (IHC) expression of Cat K. Colocalization of Cat K with CD1a, Melan-A, CD68, CD45, mast cell tryptase (MCT), and Toll-like receptors (TLRs) 4 and 9 were studied using double IHC and/or immunofluorescence (IF) staining. Expression of Cat K was also evaluated in OLP tissue samples before and after topical tacrolimus treatment. Cat K was expressed in a higher percentage of cells in the epithelial zone, and the staining intensity was stronger in the stroma in OLP compared to controls (P < 0.001). In OLP, Cat K was present mostly in melanocytes and macrophages and sporadically in basal keratinocytes, endothelial cells, and extracellularly. Cat K was found also in some fibroblasts in HC and OLP samples. Coexpression of Cat K and TLRs 4 and 9 was seen in some dendritic cells (presumably melanocytes) and macrophages. In OLP, tacrolimus treatment reduced the expression of Cat K in the epithelium but increased it in the stroma. These results suggest that Cat K is involved in the pathogenesis of OLP. Cat K possibly takes part in the modulation of matrix molecules and cellular receptors. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  18. Changes in collagenous tissue microstructures and distributions of cathepsin L in body wall of autolytic sea cucumber (Stichopus japonicus).

    Science.gov (United States)

    Liu, Yu-Xin; Zhou, Da-Yong; Ma, Dong-Dong; Liu, Yan-Fei; Li, Dong-Mei; Dong, Xiu-Ping; Tan, Ming-Qian; Du, Ming; Zhu, Bei-Wei

    2016-12-01

    The autolysis of sea cucumber (Stichopus japonicus) was induced by ultraviolet (UV) irradiation, and the changes of microstructures of collagenous tissues and distributions of cathepsin L were investigated using histological and histochemical techniques. Intact collagen fibers in fresh S. japonicus dermis were disaggregated into collagen fibrils after UV stimuli. Cathepsin L was identified inside the surface of vacuoles in the fresh S. japonicus dermis cells. After the UV stimuli, the membranes of vacuoles and cells were fused together, and cathepsin L was released from cells and diffused into tissues. The density of cathepsin L was positively correlated with the speed and degree of autolysis in different layers of body wall. Our results revealed that lysosomal cathepsin L was released from cells in response to UV stimuli, which contacts and degrades the extracellular substrates such as collagen fibers, and thus participates in the autolysis of S. japonicus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  19. Intravenous thrombolysis with recombinant tissue plasminogen activator for ischemic stroke patients over 80 years old: the Fukuoka Stroke Registry.

    Directory of Open Access Journals (Sweden)

    Ryu Matsuo

    Full Text Available The benefit of intravenous recombinant tissue plasminogen activator (rt-PA therapy for very old patients with acute ischemic stroke remains unclear. The aim of this study was to elucidate the efficacy and safety of intravenous rt-PA therapy for patients over 80 years old.Of 13,521 stroke patients registered in the Fukuoka Stroke Registry in Japan from June 1999 to February 2013, 953 ischemic stroke patients who were over 80 years old, hospitalized within 3 h of onset, and not treated with endovascular therapy were included in this study. Among them, 153 patients were treated with intravenous rt-PA (0.6 mg/kg. For propensity score (PS-matched case-control analysis, 148 patients treated with rt-PA and 148 PS-matched patients without rt-PA therapy were selected by 1:1 matching with propensity for using rt-PA. Clinical outcomes were neurological improvement, good functional outcome at discharge, in-hospital mortality, and hemorrhagic complications (any intracranial hemorrhage [ICH], symptomatic ICH, and gastrointestinal bleeding.In the full cohort of 953 patients, rt-PA use was associated positively with neurological improvement and good functional outcome, and negatively with in-hospital mortality after adjustment for multiple confounding factors. In PS-matched case-control analysis, patients treated with rt-PA were still at lower risk for unfavorable clinical outcomes than non-treated patients (neurological improvement, odds ratio 2.67, 95% confidence interval 1.61-4.40; good functional outcome, odds ratio 2.23, 95% confidence interval 1.16-4.29; in-hospital mortality, odds ratio 0.30, 95% confidence interval 0.13-0.65. There was no significant association between rt-PA use and risk of hemorrhagic complications in the full and PS-matched cohorts.Intravenous rt-PA therapy was associated with improved clinical outcomes without significant increase in risk of hemorrhagic complications in very old patients (aged>80 years with acute ischemic stroke.

  20. Baculoviral expression and characterization of human recombinant PGCP in the form of an active mature dimer and an inactive precursor protein.

    Science.gov (United States)

    Zajc, Tajana; Suban, Dejan; Rajković, Jelena; Dolenc, Iztok

    2011-02-01

    The human-blood plasma glutamate carboxypeptidase (PGCP) is a proteinase that acts on the unsubstituted N- and C-termini of dipeptides. It has been suggested that this PGCP is involved in the release of thyroxine. Furthermore, research has suggested that its activity is up-regulated in hepatitis-C-virus-infected patients with hepatocellular carcinoma. In this study expressed human PGCP in the baculovirus expression system was produced by a Sf9 insect cell line with aim to prepare sufficient amounts of active recombinant enzyme for a subsequent biological characterization. Recombinant PGCP was expressed and secreted into the medium in the form of an inactive proenzyme. It was gradually converted into an active form in the medium after three days, with the highest expression of the active form on day six. The protein was sequentially purified by a combination of various liquid chromatographies, such as hydroxyapatite, ion exchange, and gel chromatography, and as final step with affinity chromatography on Phe-Leu-Sepharose. The human PGCP was purified as an active enzyme in the dimer form and as inactive precursor protein. The dipeptidase activity was confirmed by measuring the hydrolysis of the Ser-Met dipeptide at a slightly acidic pH. Copyright © 2010 Elsevier Inc. All rights reserved.

  1. Cathepsin Gene Family Reveals Transcriptome Patterns Related to the Infective Stages of the Salmon Louse Caligus rogercresseyi.

    Directory of Open Access Journals (Sweden)

    Waleska Maldonado-Aguayo

    Full Text Available Cathepsins are proteases involved in the ability of parasites to overcome and/or modulate host defenses so as to complete their own lifecycle. However, the mechanisms underlying this ability of cathepsins are still poorly understood. One excellent model for identifying and exploring the molecular functions of cathepsins is the marine ectoparasitic copepod Caligus rogercresseyi that currently affects the Chilean salmon industry. Using high-throughput transcriptome sequencing, 56 cathepsin-like sequences were found distributed in five cysteine protease groups (B, F, L, Z, and S as well as in an aspartic protease group (D. Ontogenic transcriptome analysis evidenced that L cathepsins were the most abundant during the lifecycle, while cathepsins B and K were mostly expressed in the larval stages and adult females, thus suggesting participation in the molting processes and embryonic development, respectively. Interestingly, a variety of cathepsins from groups Z, L, D, B, K, and S were upregulated in the infective stage of copepodid, corroborating the complexity of the processes involved in the parasitic success of this copepod. Putative functional roles of cathepsins were conjectured based on the differential expressions found and on roles previously described in other phylogenetically related species. Moreover, 140 single nucleotide polymorphisms (SNP were identified in transcripts annotated for cysteine and aspartic proteases located into untranslated regions, or the coding region. This study reports for the first time the presence of cathepsin-like genes and differential expressions throughout a copepod lifecycle. The identification of cathepsins together with functional validations represents a valuable strategy for pinpointing target molecules that could be used in the development of new delousing drugs or vaccines against C. rogercresseyi.

  2. Recombinant Activated Factor VII (Eptacog Alfa Activated, NovoSeven®) in Patients with Rare Congenital Bleeding Disorders. A Systematic Review on its Use in Surgical Procedures.

    Science.gov (United States)

    Di Minno, Matteo Nicola Dario; Ambrosino, Pasquale; Myasoedova, Veronika; Amato, Manuela; Ventre, Itala; Tremoli, Elena; Minno, Alessandro Di

    2017-01-01

    In the absence of definite guidelines in the area, we have carried a systemic review to provide a thorough overview concerning the efficacy and safety of recombinant activated factor VII (rFVIIa, NovoSeven®, Novo Nordisk A/S, Bagsværd, Denmark) in patients with Glanzmann's thrombasthenia (GT) and FVII deficiency, undergoing surgical procedures. PubMed, Web of Science, Scopus and EMBASE databases was employed for the search. Three multicenter registries were identified: the Glanzmann's Thrombasthenia Registry (GTR), the Seven Treatment Evaluation Registry (STER), and a German post-marketing surveillance registry (the WIRK study). In addition, data from 10 case-series and/or single-center experiences have been summarized. We have found that the following; perioperatively, the hemostatic effectiveness of rFVIIa was high in GT patients and in those with FVII deficiency undergoing both minor and major surgical procedures. Moreover, in all studies, rFVIIa was well tolerated. Thus, the current evidence shows an optimal perioperative safety/efficacy profile of rFVIIa in the setting of these rare bleeding disorders, and provides the rationale for further studies aimed at evaluating the optimal perioperative anti-hemorrhagic prophylaxis with rFVIIa in GT and in FVII deficient patients. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Recombinant AAV-mediated in vivo long-term expression and antitumour activity of an anti-ganglioside GM3(Neu5Gc) antibody.

    Science.gov (United States)

    Piperno, G M; López-Requena, A; Predonzani, A; Dorvignit, D; Labrada, M; Zentilin, L; Burrone, O R; Cesco-Gaspere, M

    2015-12-01

    The ganglioside GM3(Neu5Gc) has gained increasing attention as therapeutic target because of its selective expression in various human tumours, such as melanoma, breast and lung cancer. 14F7 is a mouse IgG1 with specific reactivity to GM3(Neu5Gc)-positive tumours. The therapeutic activity of 14F7 has also been demonstrated in vivo, through its repetitive passive administration in tumour-bearing animals. In this work we used an alternative strategy to deliver recombinant 14F7 in vivo and analysed the therapeutic efficacy of this approach. We engineered a recombinant adeno-associated vector to direct the expression of secretable recombinant 14F7 in BALB/c animals. A single administration of the rAAV induced efficient production and secretion of the antibody in the bloodstream, with an expression level reaching plateau at ∼3 weeks after injection and persisting for almost a year. Strikingly, upon challenge with GM3(Neu5Gc)-positive X63-AG8.653 myeloma cells, tumour development was significantly delayed in animals treated with rAAV-14F7 with respect to animals treated with a control rAAV codifying for an irrelevant antibody. Finally, no significant differences in survival proportion were detected in animals injected with rAAV-14F7 or treated by standard administration of repetitive doses of purified monoclonal antibody 14F7.

  4. Expression levels of chaperones influence biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and Pseudomonas putida Baeyer-Villiger monooxygenase.

    Science.gov (United States)

    Baek, A-Hyong; Jeon, Eun-Yeong; Lee, Sun-Mee; Park, Jin-Byung

    2015-05-01

    We demonstrated for the first time that the archaeal chaperones (i.e., γ-prefoldin and thermosome) can stabilize enzyme activity in vivo. Ricinoleic acid biotransformation activity of recombinant Escherichia coli expressing Micrococcus luteus alcohol dehydrogenase and the Pseudomonas putida KT2440 Baeyer-Villiger monooxygenase improved significantly with co-expression of γ-prefoldin or recombinant themosome originating from the deep-sea hyperthermophile archaea Methanocaldococcus jannaschii. Furthermore, the degree of enhanced activity was dependent on the expression levels of the chaperones. For example, whole-cell biotransformation activity was highest at 12 µmol/g dry cells/min when γ-prefoldin expression level was approximately 46% of the theoretical maximum. This value was approximately two-fold greater than that in E. coli, where the γ-prefoldin expression level was zero or set to the theoretical maximum. Therefore, it was assumed that the expression levels of chaperones must be optimized to achieve maximum biotransformation activity in whole-cell biocatalysts. © 2014 Wiley Periodicals, Inc.

  5. New subtypes and genetic recombination in HIV type 1-infecting patients with highly active antiretroviral therapy in Peru (2008-2010).

    Science.gov (United States)

    Yabar, Carlos Augusto; Acuña, Maribel; Gazzo, Cecilia; Salinas, Gabriela; Cárdenas, Fanny; Valverde, Ada; Romero, Soledad

    2012-12-01

    HIV-1 subtype B is the most frequent strain in Peru. However, there is no available data about the genetic diversity of HIV-infected patients receiving highly active antiretroviral therapy (HAART) here. A group of 267 patients in the Peruvian National Treatment Program with virologic failure were tested for genotypic evidence of HIV drug resistance at the Instituto Nacional de Salud (INS) of Peru between March 2008 and December 2010. Viral RNA was extracted from plasma and the segments of the protease (PR) and reverse transcriptase (RT) genes were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), purified, and fully sequenced. Consensus sequences were submitted to the HIVdb Genotypic Resistance Interpretation Algorithm Database from Stanford University, and then aligned using Clustal X v.2.0 to generate a phylogenetic tree using the maximum likelihood method. Intrasubtype and intersubtype recombination analyses were performed using the SCUEAL program (Subtype Classification by Evolutionary ALgo-rithms). A total of 245 samples (91%) were successfully genotyped. The analysis obtained from the HIVdb program showed 81.5% resistance cases (n=198). The phylogenetic analysis revealed that subtype B was predominant in the population (98.8%), except for new cases of A, C, and H subtypes (n=4). Of these cases, only subtype C was imported. Likewise, recombination analysis revealed nine intersubtype and 20 intrasubtype recombinant cases. This is the first report of the presence of HIV-1 subtypes C and H in Peru. The introduction of new subtypes and circulating recombinants forms can make it difficult to distinguish resistance profiles in patients and consequently affect future treatment strategies against HIV in this country.

  6. Recombinant protein expression of Moringa oleifera lectin in methylotrophic yeast as active coagulant for sustainable high turbid water treatment.

    Science.gov (United States)

    Abd Wahid, Muhamad Azhar; Megat Mohd Noor, Megat Johari; Goto, Masafumi; Sugiura, Norio; Othman, Nor'azizi; Zakaria, Zuriati; Ahmad Mohammed, Thamer; Jusoh, Ahmad; Hara, Hirofumi

    2017-08-01

    The natural coagulant Moringa oleifera lectin (MoL) as cationic protein is a promising candidate in coagulation process of water treatment plant. Introducing the gene encoding MoL into a host, Pichia pastoris, to secrete soluble recombinant protein is assessed in this study. Initial screening using PCR confirmed the insertion of MoL gene, and SDS-PAGE analysis detected the MoL protein at 8 kDa. Cultured optimization showed the highest MoL protein at 520 mg/L was observed at 28 °C for 144 h of culturing by induction in 1% methanol. Approximately, 0.40 mg/mL of recombinant MoL protein showed 95 ± 2% turbidity removal of 1% kaolin suspension. In 0.1% kaolin suspension, the concentration of MoL at 10 μg/mL exhibits the highest turbidity reduction at 68 ± 1%. Thus, recombinant MoL protein from P. pastoris is an effective coagulant for water treatment.

  7. Inhibition of cathepsin X enzyme influences the immune response of THP-1 cells and dendritic cells infected with Helicobacter pylori

    International Nuclear Information System (INIS)

    Skvarc, Miha; Stubljar, David; Kopitar, Andreja Natasa; Jeverica, Samo; Tepes, Bojan; Kos, Janko; Ihan, Alojz

    2013-01-01

    The immune response to Helicobacter pylori importantly determines the outcome of infection as well as the success of eradication therapy. We demonstrate the role of a cysteine protease cathepsin X in the immune response to H. pylori infection. We analysed how the inhibition of cathepsin X influenced the immune response in experiments when THP-1 cells or dendritic cells isolated from patients were stimulated with 48 strains of H. pylori isolated from gastric biopsy samples of patients which had problems with the eradication of bacteria. The experiments, performed with the help of a flow cytometer, showed that the expression of Toll-like receptors (TLRs), especially TLR-4 molecules, on the membranes of THP-1 cells or dendritic cells was higher when we stimulated cells with H. pylori together with inhibitor of cathepsin X 2F12 compared to THP-1 cells or dendritic cells stimulated with H. pylori only, and also in comparison with negative control samples. We also demonstrated that when we inhibited the action of cathepsin X in THP-1 cells, the concentrations of pro-inflammatory cytokines were lower than when THP-1 cell were stimulated with H. pylori only. We demonstrated that inhibition of cathepsin X influences the internalization of TLR-2 and TLR-4. TLR-2 and TLR-4 redistribution to intra-cytoplasmic compartments is hampered if cathepsin X is blocked. The beginning of a successful immune response against H. pylori in the case of inhibition of cathepsin X is delayed

  8. Fasciola gigantica cathepsin L proteinase-based synthetic peptide for immunodiagnosis and prevention of sheep fasciolosis

    Czech Academy of Sciences Publication Activity Database

    Ježek, Jan; El Ridi, R.; Salah, M.; Wagih, A.; Aziz, H. W.; Tallima, H.; El Shafie, M. H.; Khalek, T. A.; Ammou, F. F. A.; Strongylis, C.; Moussis, V.; Tsikaris, V.

    2008-01-01

    Roč. 90, č. 3 (2008), s. 349-357 ISSN 0006-3525 Institutional research plan: CEZ:AV0Z40550506 Keywords : cathepsin L proteinase * peptides * sequential oligopeptide carriers * synthetic peptide vaccine * Fasciiola gigantica Subject RIV: CC - Organic Chemistry Impact factor: 2.823, year: 2008

  9. Prognostic and predictive value of cathepsin X in serum from colorectal cancer patients

    DEFF Research Database (Denmark)

    Vižin, Tjaša; Christensen, Ib Jarle; Wilhelmsen, Michael

    2014-01-01

    , but for patients in stages I-III with local resectable disease. The significant association of cathepsin X with survival in a group of patients who received no chemotherapy and the absence of this association in the group who received chemotherapy, suggest the possible predictive value for response to chemotherapy...

  10. Oestrogen regulates the expression of cathepsin E-A-like gene ...

    Indian Academy of Sciences (India)

    Hang Zheng

    2018-02-28

    Feb 28, 2018 ... 1College of Animal Science and Veterinary Medicine, Henan Agricultural .... evaluated the expression regulation mechanism of the gene ... C with ad libitum water and food. ... embryonic liver following the method previously described .... Cloning and sequence analysis of chicken cathepsin E-A-like gene.

  11. Cathepsin L is crucial for the development of early experimental diabetic nephropathy

    NARCIS (Netherlands)

    Garsen, M.; Rops, A.; Dijkman, H.B.; Willemsen, B.K.; Kuppevelt, T.H. van; Russel, F.G.M.; Rabelink, T.J.; Berden, J.H.; Reinheckel, T.; Vlag, J. van der

    2016-01-01

    Proteinuria is one of the first clinical signs of diabetic nephropathy and an independent predictor for the progression to renal failure. Cathepsin L, a lysosomal cysteine protease, can be involved in the development of proteinuria by degradation of proteins that are important for normal podocyte

  12. Parasite Cathepsin D-Like Peptidases and Their Relevance as Therapeutic Targets

    Czech Academy of Sciences Publication Activity Database

    Sojka, D.; Hartmann, D.; Bartošová-Sojková, P.; Dvořák, Jan

    2016-01-01

    Roč. 32, č. 9 (2016), s. 708-723 ISSN 1471-4922 R&D Projects: GA ČR GA13-11043S; GA ČR(CZ) GAP302/11/1481 Institutional support: RVO:61388963 Keywords : aspartic peptidases * cathepsin D * hemoglobinolysis * parasites * vectors Subject RIV: CE - Biochemistry Impact factor: 6.333, year: 2016

  13. Recombinant production of a chimeric antimicrobial peptide in E. coli and assessment of its activity against some avian clinically isolated pathogens.

    Science.gov (United States)

    Tanhaiean, Abass; Azghandi, Marjan; Razmyar, Jamshid; Mohammadi, Elyas; Sekhavati, Mohammad Hadi

    2018-06-08

    Over the last decades, poultry industry faced to the rapid emergence of multidrug-resistant bacteria as a global concern. Antimicrobial peptide (AMPs) known as potential antibiotic alternative and were considered as a new antimicrobial agent. Current methods of production and purification of AMPs have several limitations such as: costly, time-consuming and killing the producing host cells in recombinant form. In the present study, a chimeric peptide derived from camel lactoferrin was produced in Escherichia coli periplasmic space using a pET-based expression system and its antibacterial activity was determined on some avian pathogens in vitro. A carboxy-terminal polyhistidine tag was used for purification by Ni 2+ affinity chromatography with an average yield of 0.42 g/L. The His-tagged chimeric peptide showed different range of antimicrobial activity against clinically isolated avian pathogens with low chicken blood hemolysis activity and high serum stability. Overall, the results of this investigation showed the recombinant chimeric peptide was successfully expressed in pET-based expression system and could be considered as a proper alternative for some currently used antibiotics in poultry industry and drugs veterinary medicine. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity

    Energy Technology Data Exchange (ETDEWEB)

    Jessop, Forrest; Hamilton, Raymond F.; Rhoderick, Joseph F.; Fletcher, Paige; Holian, Andrij, E-mail: andrij.holian@umontana.edu

    2017-03-01

    NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle-induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi-walled carbon nanotubes, and titanium nanobelts. Bafilomycin A1 treatment of particle-exposed macrophages also resulted in decreased active cathepsin L in the cytosol, a surrogate measure for leaked cathepsin B, which was associated with less NLRP3 inflammasome activity. Silica-induced LMP was partially dependent upon lysosomal cathepsins B and L, whereas nanoparticle-induced LMP occurred independent of cathepsin activity. Furthermore, inhibition of lysosomal cathepsin activity with CA-074-Me decreased the release of High Mobility Group Box 1. Together, these data support the notion that lysosome acidification is a prerequisite for particle-induced LMP, and the resultant leak of lysosome cathepsins is a primary regulator of ongoing NLRP3 inflammasome activity and release of HMGB1. - Highlights: • Silica and nanoparticles cause LMP in macrophages in vitro and in vivo. • Phagolysosome acidification is required for particle-induced LMP. • Cathepsin B and L are not required for nanoparticle-induced LMP. • Cathepsin B/L regulate the secretion of HMGB1 with particle exposure.

  15. Phagolysosome acidification is required for silica and engineered nanoparticle-induced lysosome membrane permeabilization and resultant NLRP3 inflammasome activity

    International Nuclear Information System (INIS)

    Jessop, Forrest; Hamilton, Raymond F.; Rhoderick, Joseph F.; Fletcher, Paige; Holian, Andrij

    2017-01-01

    NLRP3 inflammasome activation occurs in response to hazardous particle exposures and is critical for the development of particle-induced lung disease. Mechanisms of Lysosome Membrane Permeabilization (LMP), a central pathway for activation of the NLRP3 inflammasome by inhaled particles, are not fully understood. We demonstrate that the lysosomal vATPases inhibitor Bafilomycin A1 blocked LMP in vitro and ex vivo in primary murine macrophages following exposure to silica, multi-walled carbon nanotubes, and titanium nanobelts. Bafilomycin A1 treatment of particle-exposed macrophages also resulted in decreased active cathepsin L in the cytosol, a surrogate measure for leaked cathepsin B, which was associated with less NLRP3 inflammasome activity. Silica-induced LMP was partially dependent upon lysosomal cathepsins B and L, whereas nanoparticle-induced LMP occurred independent of cathepsin activity. Furthermore, inhibition of lysosomal cathepsin activity with CA-074-Me decreased the release of High Mobility Group Box 1. Together, these data support the notion that lysosome acidification is a prerequisite for particle-induced LMP, and the resultant leak of lysosome cathepsins is a primary regulator of ongoing NLRP3 inflammasome activity and release of HMGB1. - Highlights: • Silica and nanoparticles cause LMP in macrophages in vitro and in vivo. • Phagolysosome acidification is required for particle-induced LMP. • Cathepsin B and L are not required for nanoparticle-induced LMP. • Cathepsin B/L regulate the secretion of HMGB1 with particle exposure.

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

    Science.gov (United States)

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

    2014-08-01

    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. Recombinant activated factor VII in the treatment of bleeds and for the prevention of surgery-related bleeding in congenital haemophilia with inhibitors.

    Science.gov (United States)

    Santagostino, Elena; Escobar, Miguel; Ozelo, Margareth; Solimeno, Luigi; Arkhammar, Per; Lee, Hye Youn; Rosu, Gabriela; Giangrande, Paul

    2015-06-01

    The availability of recombinant activated factor VII (rFVIIa, eptacog alfa activated) has greatly advanced the care of patients with haemophilia A or B who have developed inhibitors against the infused replacement factor. Recombinant FVIIa is licensed for the on-demand treatment of bleeding episodes and the prevention of bleeding in surgery or invasive procedures in patients with congenital haemophilia with inhibitors. This article attempts to review in detail the extensive evidence of rFVIIa in congenital haemophilia patients with inhibitors. Patients with acute bleeding episodes are best treated on demand at home, to achieve the short- and long-term benefits of rapid bleed control. Key prospective studies have shown that rFVIIa achieves consistently high efficacy rates in the management of acute (including joint) bleeds in inhibitor patients in the home treatment setting. Substantial post-approval data from key registries also support the on-demand efficacy profile of rFVIIa established by the prospective clinical trials. The availability of rFVIIa has allowed major surgery to become a reality for inhibitor patients. Studies in key surgery, including orthopaedic procedures, have found that rFVIIa provides consistently high efficacy rates. Importantly, the wealth of data does not raise any unexpected safety concerns surrounding rFVIIa use; this is likely because rFVIIa is a recombinant product with a localised mechanism of action at the site of vascular injury. In summary, rFVIIa is established as an effective and well-tolerated first-line treatment for on-demand bleeding control and bleed prevention during minor and major (including elective orthopaedic) surgery in inhibitor patients. Use of rFVIIa has been a major step towards narrowing the gap in outcomes between inhibitor patients and non-inhibitor patients. Copyright © 2015 Elsevier Ltd. All rights reserved.

  18. Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults.

    Science.gov (United States)

    Szpakowski, Piotr; Biet, Franck; Locht, Camille; Paszkiewicz, Małgorzata; Rudnicka, Wiesława; Druszczyńska, Magdalena; Allain, Fabrice; Fol, Marek; Pestel, Joël; Kowalewicz-Kulbat, Magdalena

    2015-01-01

    Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG), the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18) and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4(+) T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

  19. Dendritic Cell Activity Driven by Recombinant Mycobacterium bovis BCG Producing Human IL-18, in Healthy BCG Vaccinated Adults

    Directory of Open Access Journals (Sweden)

    Piotr Szpakowski

    2015-01-01

    Full Text Available Tuberculosis remains an enormous global burden, despite wide vaccination coverage with the Bacillus Calmette-Guérin (BCG, the only vaccine available against this disease, indicating that BCG-driven immunity is insufficient to protect the human population against tuberculosis. In this study we constructed recombinant BCG producing human IL-18 (rBCGhIL-18 and investigated whether human IL-18 produced by rBCGhIL-18 modulates DC functions and enhances Th1 responses to mycobacterial antigens in humans. We found that the costimulatory CD86 and CD80 molecules were significantly upregulated on rBCGhIL-18-infected DCs, whereas the stimulation of DCs with nonrecombinant BCG was less effective. In contrast, both BCG strains decreased the DC-SIGN expression on human DCs. The rBCGhIL-18 increased IL-23, IL-10, and IP-10 production by DCs to a greater extent than nonrecombinant BCG. In a coculture system of CD4+ T cells and loaded DCs, rBCGhIL-18 favoured strong IFN-γ but also IL-10 production by naive T cells but not by memory T cells. This was much less the case for nonrecombinant BCG. Thus the expression of IL-18 by recombinant BCG increases IL-23, IP-10, and IL-10 expression by human DCs and enhances their ability to induce IFN-γ and IL-10 expression by naive T cells, without affecting the maturation phenotype of the DCs.

  20. 177Lu-labeled HPMA copolymers utilizing cathepsin B and S cleavable linkers: Synthesis, characterization and preliminary in vivo investigation in a pancreatic cancer model

    International Nuclear Information System (INIS)

    Ogbomo, Sunny M.; Shi, Wen; Wagh, Nilesh K.; Zhou, Zhengyuan; Brusnahan, Susan K.; Garrison, Jered C.

    2013-01-01

    Introduction: A major barrier to the advancement of therapeutic nanomedicines has been the non-target toxicity caused by the accumulation of the drug delivery systems in organs associated with the reticuloendothelial system, particularly the liver and spleen. Herein, we report the development of peptide based metabolically active linkers (MALs) that are enzymatically cleaved by cysteine cathepsin B and S, two proteases highly expressed in the liver and spleen. The overall goal of this approach is to utilize the MALs to lower the non-target retention and toxicity of radiolabeled drug delivery systems, thus resulting in higher diagnostic and radiotherapeutic efficacy. Methods: In this study three MALs (MAL0, MAL1 and MAL2) were investigated. MAL1 and MAL2 are composed of known substrates of cathepsin B and S, respectively, while MAL0 is a non-cleavable control. Both MAL1 and MAL2 were shown to undergo enzymatic cleavage with the appropriate cathepsin protease. Subsequent to conjugation to the HPMA copolymer and radiolabeling with 177 Lu, the peptide–polymer conjugates were renamed 177 Lu-metabolically active copolymers ( 177 Lu-MACs) with the corresponding designations: 177 Lu-MAC0, 177 Lu-MAC1 and 177 Lu-MAC2. Results: In vivo evaluation of the 177 Lu-MACs was performed in an HPAC human pancreatic cancer xenograft mouse model. 177 Lu-MAC1 and 177 Lu-MAC2 demonstrated 3.1 and 2.1 fold lower liver retention, respectively, compared to control ( 177 Lu-MAC0) at 72 h post-injection. With regard to spleen retention, 177 Lu-MAC1 and 177 Lu-MAC2 each exhibited a nearly fourfold lower retention, relative to control, at the 72 h time point. However, the tumor accumulation of the 177 Lu-MAC0 was two to three times greater than 177 Lu-MAC1 and 177 Lu-MAC2 at the same time point. The MAL approach demonstrated the capability of substantially reducing the non-target retention of the 177 Lu-labeled HPMA copolymers. Conclusions: While further studies are needed to optimize the

  1. Substrate-derived triazolo- and azapeptides as inhibitors of cathepsins K and S.

    Science.gov (United States)

    Galibert, Matthieu; Wartenberg, Mylène; Lecaille, Fabien; Saidi, Ahlame; Mavel, Sylvie; Joulin-Giet, Alix; Korkmaz, Brice; Brömme, Dieter; Aucagne, Vincent; Delmas, Agnès F; Lalmanach, Gilles

    2018-01-20

    Cathepsin (Cat) K is a critical bone-resorbing protease and is a relevant target for the treatment of osteoporosis and bone metastasis, while CatS is an attractive target for drugs in autoimmune diseases (e.g. rheumatoid arthritis), emphysema or neuropathic pain. Despite major achievements, current pharmacological inhibitors are still lacking in safety and may have damaging side effects. A promising strategy for developing safer reversible and competitive inhibitors as new lead compounds could be to insert non-cleavable bonds at the scissile P1-P1' position of selective substrates of CatS and CatK. Accordingly, we introduced a 1,4-disubstituted 1,2,3-triazole heterocycle that mimics most of the features of a trans-amide bond, or we incorporated a semicarbazide bond (azaGly residue) by replacing the α-carbon of the glycyl residue at P1 by a nitrogen atom. AzaGly-containing peptidomimetics inhibited powerfully their respective target proteases in the nM range, while triazolopeptides were weaker inhibitors (Ki in the μM range). The selectivity of the azaGly CatS inhibitor (1b) was confirmed by using spleen lysates from wild-type vs CatS-deficient mice. Alternatively, the azaGly bradykinin-derived CatK inhibitor (2b) potently inhibited CatK (Ki = 9 nM) and impaired its kininase activity in vitro. Molecular modeling studies support that the semicarbazide bond of 2b is more favorable than the 1,2,3-triazole linkage of the bradykinin-derived pseudopeptide 2a to preserve an effective affinity towards CatK, its protease target. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  2. Enhancement of 2,3-butanediol production from Jerusalem artichoke tuber extract by a recombinant Bacillus sp. strain BRC1 with increased inulinase activity.

    Science.gov (United States)

    Park, Jang Min; Oh, Baek-Rock; Kang, In Yeong; Heo, Sun-Yeon; Seo, Jeong-Woo; Park, Seung-Moon; Hong, Won-Kyung; Kim, Chul Ho

    2017-07-01

    A Bacillus sp. strain named BRC1 is capable of producing 2,3-butanediol (2,3-BD) using hydrolysates of the Jerusalem artichoke tuber (JAT), a rich source of the fructose polymer inulin. To enhance 2,3-BD production, we undertook an extensive analysis of the Bacillus sp. BRC1 genome, identifying a putative gene (sacC) encoding a fructan hydrolysis enzyme and characterizing the activity of the resulting recombinant protein expressed in and purified from Escherichia coli. Introduction of the sacC gene into Bacillus sp. BRC1 using an expression vector increased enzymatic activity more than twofold. Consistent with this increased enzyme expression, 2,3-BD production from JAT was also increased from 3.98 to 8.10 g L -1 . Fed-batch fermentation of the recombinant strain produced a maximal level of 2,3-BD production of 28.6 g L -1 , showing a high theoretical yield of 92.3%.

  3. Recombinant Collagenlike Proteins

    Science.gov (United States)

    Fertala, Andzej

    2007-01-01

    A group of collagenlike recombinant proteins containing high densities of biologically active sites has been invented. The method used to express these proteins is similar to a method of expressing recombinant procollagens and collagens described in U. S. Patent 5,593,859, "Synthesis of human procollagens and collagens in recombinant DNA systems." Customized collagenous proteins are needed for biomedical applications. In particular, fibrillar collagens are attractive for production of matrices needed for tissue engineering and drug delivery. Prior to this invention, there was no way of producing customized collagenous proteins for these and other applications. Heretofore, collagenous proteins have been produced by use of such biological systems as yeasts, bacteria, and transgenic animals and plants. These products are normal collagens that can also be extracted from such sources as tendons, bones, and hides. These products cannot be made to consist only of biologically active, specific amino acid sequences that may be needed for specific applications. Prior to this invention, it had been established that fibrillar collagens consist of domains that are responsible for such processes as interaction with cells, binding of growth factors, and interaction with a number of structural proteins present in the extracellular matrix. A normal collagen consists of a sequence of domains that can be represented by a corresponding sequence of labels, e.g., D1D2D3D4. A collagenlike protein of the present invention contains regions of collagen II that contain multiples of a single domain (e.g., D1D1D1D1 or D4D4D4D4) chosen for its specific biological activity. By virtue of the multiplicity of the chosen domain, the density of sites having that specific biological activity is greater than it is in a normal collagen. A collagenlike protein according to this invention can thus be made to have properties that are necessary for tissue engineering.

  4. A successful strategy for the recovering of active P21, an insoluble recombinant protein of Trypanosoma cruzi

    Science.gov (United States)

    Santos, Marlus Alves Dos; Teixeira, Francesco Brugnera; Moreira, Heline Hellen Teixeira; Rodrigues, Adele Aud; Machado, Fabrício Castro; Clemente, Tatiana Mordente; Brigido, Paula Cristina; Silva, Rebecca Tavares E.; Purcino, Cecílio; Gomes, Rafael Gonçalves Barbosa; Bahia, Diana; Mortara, Renato Arruda; Munte, Claudia Elisabeth; Horjales, Eduardo; da Silva, Claudio Vieira

    2014-03-01

    Structural studies of proteins normally require large quantities of pure material that can only be obtained through heterologous expression systems and recombinant technique. In these procedures, large amounts of expressed protein are often found in the insoluble fraction, making protein purification from the soluble fraction inefficient, laborious, and costly. Usually, protein refolding is avoided due to a lack of experimental assays that can validate correct folding and that can compare the conformational population to that of the soluble fraction. Herein, we propose a validation method using simple and rapid 1D 1H nuclear magnetic resonance (NMR) spectra that can efficiently compare protein samples, including individual information of the environment of each proton in the structure.

  5. Effect of hydrophilicity of carbon nanotube arrays on the release rate and activity of recombinant human bone morphogenetic protein-2

    Energy Technology Data Exchange (ETDEWEB)

    Han Zhaojun; Ostrikov, Kostya [Plasma Nanoscience Centre Australia (PNCA), CSIRO Materials Science and Engineering, Lindfield, New South Wales 2070 (Australia); Tan, Cher Ming; Tay, Beng Kang [School of Electrical and Electronic Engineering, Nanyang Technological University, 639798 (Singapore); Peel, Sean A F, E-mail: zhaojun.han@csiro.au [Department of Dentistry, University of Toronto, Toronto, ON, M5G 1G6 (Canada)

    2011-07-22

    Novel nanostructures such as vertically aligned carbon nanotube (CNT) arrays have received increasing interest as drug delivery carriers. In the present study, two CNT arrays with extreme surface wettabilities are fabricated and their effects on the release of recombinant human bone morphogenetic protein-2 (rhBMP-2) are investigated. It is found that the superhydrophilic arrays retained a larger amount of rhBMP-2 than the superhydrophobic ones. Further use of a poloxamer diffusion layer delayed the initial burst and resulted in a greater total amount of rhBMP-2 released from both surfaces. In addition, rhBMP-2 bound to the superhydrophilic CNT arrays remained bioactive while they denatured on the superhydrophobic surfaces. These results are related to the combined effects of rhBMP-2 molecules interacting with poloxamer and the surface, which could be essential in the development of advanced carriers with tailored surface functionalities.

  6. Dependence of Immunoglobulin Class Switch Recombination in B Cells on Vesicular Release of ATP and CD73 Ectonucleotidase Activity

    Directory of Open Access Journals (Sweden)

    Francesca Schena

    2013-06-01

    Full Text Available Immunoglobulin (Ig isotype diversification by class switch recombination (CSR is an essential process for mounting a protective humoral immune response. Ig CSR deficiencies in humans can result from an intrinsic B cell defect; however, most of these deficiencies are still molecularly undefined and diagnosed as common variable immunodeficiency (CVID. Here, we show that extracellular adenosine critically contributes to CSR in human naive and IgM memory B cells. In these cells, coordinate stimulation of B cell receptor and toll-like receptors results in the release of ATP stored in Ca2+-sensitive secretory vesicles. Plasma membrane ectonucleoside triphosphate diphosphohydrolase 1 CD39 and ecto-5′-nucleotidase CD73 hydrolyze ATP to adenosine, which induces CSR in B cells in an autonomous fashion. Notably, CVID patients with impaired class-switched antibody responses are selectively deficient in CD73 expression in B cells, suggesting that CD73-dependent adenosine generation contributes to the pathogenesis of this disease.

  7. Cathepsin B is up-regulated and mediates extracellular matrix degradation in trabecular meshwork cells following phagocytic challenge.

    Directory of Open Access Journals (Sweden)

    Kristine Porter

    Full Text Available Cells in the trabecular meshwork (TM, a tissue responsible for draining aqueous humor out of the eye, are known to be highly phagocytic. Phagocytic activity in TM cells is thought to play an important role in outflow pathway physiology. However, the molecular mechanisms triggered by phagocytosis in TM cells are unknown. Here we investigated the effects of chronic phagocytic stress on lysosomal function using different phagocytic ligands (E. coli, carboxylated beads, collagen I-coated beads, and pigment. Lysotracker red co-localization and electron micrographs showed the maturation of E. coli- and collagen I-coated beads-containing phagosomes into phagolysosomes. Maturation of phagosomes into phagolysosomes was not observed with carboxylated beads or pigment particles. In addition, phagocytosis of E. coli and collagen I-coated beads led to increased lysosomal mass, and the specific up-regulation and activity of cathepsin B (CTSB. Higher levels of membrane-bound and secreted CTSB were also detected. Moreover, in vivo zymography showed the intralysosomal degradation of ECM components associated with active CTSB, as well as an overall increased gelatinolytic activity in phagocytically challenged TM cells. This increased gelatinolytic activity with phagocytosis was partially blocked with an intracellular CTSB inhibitor. Altogether, these results suggest a potential role of phagocytosis in outflow pathway tissue homeostasis through the up-regulation and/or proteolytic activation of extracellular matrix remodeling genes.

  8. Critical appraisal of the role of recombinant activated factor VII in the treatment of hemophilia patients with inhibitors