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Sample records for human aspartic protease

  1. Secreted aspartic proteases are not required for invasion of reconstituted human epithelia by Candida albicans.

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    Lermann, Ulrich; Morschhäuser, Joachim

    2008-11-01

    A well-known virulence attribute of the human-pathogenic yeast Candida albicans is the secretion of aspartic proteases (Saps), which may contribute to colonization and infection of different host niches by degrading tissue barriers, destroying host defence molecules, or digesting proteins for nutrient supply. The role of individual Sap isoenzymes, which are encoded by a large gene family, for the pathogenicity of C. albicans has been investigated by assessing the virulence of mutants lacking specific SAP genes and by studying the expression pattern of the SAP genes in various models of superficial and systemic infections. We used a recombination-based genetic reporter system to detect the induction of the SAP1-SAP6 genes during infection of reconstituted human vaginal epithelium. Only SAP5, but none of the other tested SAP genes, was detectably activated in this in vitro infection model. To directly address the importance of the SAP1-SAP6 genes for invasion of reconstituted human epithelia (RHE), we constructed a set of mutants of the wild-type C. albicans model strain SC5314 in which either single or multiple SAP genes were specifically deleted. Even mutants lacking all of the SAP1-SAP3 or the SAP4-SAP6 genes displayed the same capacity to invade and damage both oral and vaginal RHE as their wild-type parental strain, in contrast to a nonfilamentous efg1Delta mutant that was avirulent under these conditions. We therefore conclude from these results that the secreted aspartic proteases Sap1p-Sap6p are not required for invasion of RHE by C. albicans.

  2. Dataset of cocoa aspartic protease cleavage sites

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    Katharina Janek

    2016-09-01

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

  3. Potent radiolabeled human renin inhibitor, [3H]SR42128: enzymatic, kinetic, and binding studies to renin and other aspartic proteases

    International Nuclear Information System (INIS)

    Cumin, F.; Nisato, D.; Gagnol, J.P.; Corvol, P.

    1987-01-01

    The in vitro binding of [ 3 H]SR42128 (Iva-Phe-Nle-Sta-Ala-Sta-Arg), a potent inhibitor of human renin activity, to purified human renin and a number of other aspartic proteases was examined. SR42128 was found to be a competitive inhibitor of human renin, with a K/sub i/ of 0.35 nM at pH 5.7 and 2.0 nM at pH 7.4; it was thus more effective at pH 5.7 than at pH 7.4. Scatchard analysis of the interaction binding of [ 3 H]SR42128 to human renin indicated that binding was reversible and saturable at both pH 5.7 and pH 7.4. There was a single class of binding sites, and the K/sub D/ was 0.9 nM at pH 5.7 and 1 nM at pH 7.4. The association rate was 10 times more rapid at pH 5.7 than at pH 7.4, but there was no difference between the rates of dissociation of the enzyme-inhibitor complex at the two pHs. The effect of pH on the binding of [ 3 H]SR42128 to human renin, cathepsin D, pepsin, and gastricsin was also examined over the pH range 3-8. All the aspartic proteases had a high affinity for the inhibitor at low pH. However, at pH 7.4, [ 3 H]SR42128 was bound only to human renin and to none of the other aspartic proteases. Competitive binding studies with [ 3 H]SR42128 and a number of other inhibitors on human renin or cathepsin D were used to examine the relationships between structure and activity in these systems. The study as a whole indicates that pH plays a major role in the binding of [ 3 H]SR42128 to aspartic proteases and that the nature of the inhibitor residue reacting with the renin S 2 subsites is of critical importance for the specificity of the renin-inhibitor interaction

  4. Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

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    Díaz, M E; Rocha, G F; Kise, F; Rosso, A M; Guevara, M G; Parisi, M G

    2018-05-08

    Plant proteases play a fundamental role in several processes like growth, development and in response to biotic and abiotic stress. In particular, aspartic proteases (AP) are expressed in different plant organs and have antimicrobial activity. Previously, we purified an AP from Salpichroa origanifolia fruits called salpichroin. The aim of this work was to determine the cytotoxic activity of this enzyme on selected plant and human pathogens. For this purpose, the growth of the selected pathogens was analysed after exposure to different concentrations of salpichroin. The results showed that the enzyme was capable of inhibiting Fusarium solani and Staphylococcus aureus in a dose-dependent manner. It was determined that 1·2 μmol l -1 of salpichroin was necessary to inhibit 50% of conidial germination, and the minimal bactericidal concentration was between 1·9 and 2·5 μmol l -1 . Using SYTOX Green dye we were able to demonstrate that salpichroin cause membrane permeabilization. Moreover, the enzyme treated with its specific inhibitor pepstatin A did not lose its antibacterial activity. This finding demonstrates that the cytotoxic activity of salpichroin is due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of the AP could represent a potential alternative for the control of pathogens that affect humans or crops of economic interest. This study provides insights into the antimicrobial activity of an aspartic protease isolated from Salpichroa origanifolia fruits on plant and human pathogens. The proteinase inhibited Fusarium solani and Staphylococcus aureus in a dose-dependent manner due to the alteration of the cell plasma membrane barrier but not due to its proteolytic activity. Antimicrobial activity of salpichroin suggests its potential applications as an important tool for the control of pathogenic micro-organisms affecting humans and crops of economic interest. Therefore, it would

  5. Aspartic protease activities of schistosomes cleave mammalian hemoglobins in a host-specific manner

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    Jeffrey W Koehler

    2007-02-01

    Full Text Available We examined the efficiency of digestion of hemoglobin from four mammalian species, human, cow, sheep, and horse by acidic extracts of mixed sex adults of Schistosoma japonicum and S. mansoni. Activity ascribable to aspartic protease(s from S. japonicum and S. mansoni cleaved human hemoglobin. In addition, aspartic protease activities from S. japonicum cleaved hemoglobin from bovine, sheep, and horse blood more efficiently than did the activity from extracts of S. mansoni. These findings support the hypothesis that substrate specificity of hemoglobin-degrading proteases employed by blood feeding helminth parasites influences parasite host species range; differences in amino acid sequences in key sites of the parasite proteases interact less or more efficiently with the hemoglobins of permissive or non-permissive hosts.

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

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    Kernaghan, Gavin; Mayerhofer, Michael

    2017-01-01

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

  7. Expression, purification, and characterization of the Necator americanus aspartic protease-1 (Na-APR-1 (M74)) antigen, a component of the bivalent human hookworm vaccine.

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    Seid, Christopher A; Curti, Elena; Jones, R Mark; Hudspeth, Elissa; Rezende, Wanderson; Pollet, Jeroen; Center, Lori; Versteeg, Leroy; Pritchard, Sonya; Musiychuk, Konstantin; Yusibov, Vidadi; Hotez, Peter J; Bottazzi, Maria Elena

    2015-01-01

    Over 400 million people living in the world's poorest developing nations are infected with hookworms, mostly of the genus Necator americanus. A bivalent human hookworm vaccine composed of the Necator americanus Glutathione S-Transferase-1 (Na-GST-1) and the Necator americanus Aspartic Protease-1 (Na-APR-1 (M74)) is currently under development by the Sabin Vaccine Institute Product Development Partnership (Sabin PDP). Both monovalent vaccines are currently in Phase 1 trials. Both Na-GST-1 and Na-APR-1 antigens are expressed as recombinant proteins. While Na-GST-1 was found to express with high yields in Pichia pastoris, the level of expression of Na-APR-1 in this host was too low to be suitable for a manufacturing process. When the tobacco plant Nicotiana benthamiana was evaluated as an expression system, acceptable levels of solubility, yield, and stability were attained. Observed expression levels of Na-APR-1 (M74) using this system are ∼300 mg/kg. Here we describe the achievements and obstacles encountered during process development as well as characterization and stability of the purified Na-APR-1 (M74) protein and formulated vaccine. The expression, purification and analysis of purified Na-APR-1 (M74) protein obtained from representative 5 kg reproducibility runs performed to qualify the Na-APR-1 (M74) production process is also presented. This process has been successfully transferred to a pilot plant and a 50 kg scale manufacturing campaign under current Good Manufacturing Practice (cGMP) has been performed. The 50 kg run has provided a sufficient amount of protein to support the ongoing hookworm vaccine development program of the Sabin PDP.

  8. RC1339/APRc from Rickettsia conorii is a novel aspartic protease with properties of retropepsin-like enzymes.

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    Rui Cruz

    2014-08-01

    Full Text Available Members of the species Rickettsia are obligate intracellular, gram-negative, arthropod-borne pathogens of humans and other mammals. The life-threatening character of diseases caused by many Rickettsia species and the lack of reliable protective vaccine against rickettsioses strengthens the importance of identifying new protein factors for the potential development of innovative therapeutic tools. Herein, we report the identification and characterization of a novel membrane-embedded retropepsin-like homologue, highly conserved in 55 Rickettsia genomes. Using R. conorii gene homologue RC1339 as our working model, we demonstrate that, despite the low overall sequence similarity to retropepsins, the gene product of rc1339 APRc (for Aspartic Protease from Rickettsia conorii is an active enzyme with features highly reminiscent of this family of aspartic proteases, such as autolytic activity impaired by mutation of the catalytic aspartate, accumulation in the dimeric form, optimal activity at pH 6, and inhibition by specific HIV-1 protease inhibitors. Moreover, specificity preferences determined by a high-throughput profiling approach confirmed common preferences between this novel rickettsial enzyme and other aspartic proteases, both retropepsins and pepsin-like. This is the first report on a retropepsin-like protease in gram-negative intracellular bacteria such as Rickettsia, contributing to the analysis of the evolutionary relationships between the two types of aspartic proteases. Additionally, we have also shown that APRc is transcribed and translated in R. conorii and R. rickettsii and is integrated into the outer membrane of both species. Finally, we demonstrated that APRc is sufficient to catalyze the in vitro processing of two conserved high molecular weight autotransporter adhesin/invasion proteins, Sca5/OmpB and Sca0/OmpA, thereby suggesting the participation of this enzyme in a relevant proteolytic pathway in rickettsial life-cycle. As a

  9. Molecular characterization of 45 kDa aspartic protease of Trichinella spiralis.

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

    2012-12-21

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

  10. Discovery of MK-8718, an HIV Protease Inhibitor Containing a Novel Morpholine Aspartate Binding Group

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    Bungard, Christopher J.; Williams, Peter D.; Ballard, Jeanine E.; Bennett, David J.; Beaulieu, Christian; Bahnck-Teets, Carolyn; Carroll, Steve S.; Chang, Ronald K.; Dubost, David C.; Fay, John F.; Diamond, Tracy L.; Greshock, Thomas J.; Hao, Li; Holloway, M. Katharine; Felock, Peter J.; Gesell, Jennifer J.; Su, Hua-Poo; Manikowski, Jesse J.; McKay, Daniel J.; Miller, Mike; Min, Xu; Molinaro, Carmela; Moradei, Oscar M.; Nantermet, Philippe G.; Nadeau, Christian; Sanchez, Rosa I.; Satyanarayana, Tummanapalli; Shipe, William D.; Singh, Sanjay K.; Truong, Vouy Linh; Vijayasaradhi, Sivalenka; Wiscount, Catherine M.; Vacca, Joseph P.; Crane, Sheldon N.; McCauley, John A. (Merck); (Albany MR)

    2016-07-14

    A novel HIV protease inhibitor was designed using a morpholine core as the aspartate binding group. Analysis of the crystal structure of the initial lead bound to HIV protease enabled optimization of enzyme potency and antiviral activity. This afforded a series of potent orally bioavailable inhibitors of which MK-8718 was identified as a compound with a favorable overall profile.

  11. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis

    Czech Academy of Sciences Publication Activity Database

    Kádek, Alan; Tretyachenko, V.; Mrázek, Hynek; Ivanova, Ljubina; Halada, Petr; Rey, M.; Schriemer, D. C.; Man, Petr

    2014-01-01

    Roč. 95, MAR 2014 (2014), s. 121-128 ISSN 1046-5928 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : Plant aspartic protease * Nepenthesin * Protease characterization Subject RIV: CE - Biochemistry Impact factor: 1.695, year: 2014

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

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

    2016-08-01

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

  13. Atomic resolution crystal structure of Sapp2p, a secreted aspartic protease from Candida parapsilosis

    Czech Academy of Sciences Publication Activity Database

    Dostál, Jiří; Pecina, Adam; Hrušková-Heidingsfeldová, Olga; Marečková, L.; Pichová, Iva; Řezáčová, Pavlína; Lepšík, Martin; Brynda, Jiří

    2015-01-01

    Roč. 71, č. 12 (2015), s. 2494-2504 ISSN 1399-0047 R&D Projects: GA ČR(CZ) GA14-23022S Institutional support: RVO:61388963 ; RVO:68378050 Keywords : aspartic protease * Candida parapsilosis * Sapp2p * crystal structure * ultrahigh resolution * interaction energy * quantum mechanics Subject RIV: CE - Biochemistry; EB - Genetics ; Molecular Biology (UMG-J) Impact factor: 2.674, year: 2014

  14. Production, purification and characterization of an aspartic protease from Aspergillus foetidus.

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    Souza, Paula Monteiro; Werneck, Gabriela; Aliakbarian, Bahar; Siqueira, Felix; Ferreira Filho, Edivaldo Ximenes; Perego, Patrizia; Converti, Attilio; Magalhães, Pérola Oliveira; Junior, Adalberto Pessoa

    2017-11-01

    An acidic thermostable protease was extracellularly produced either in shake flask or in stirred tank bioreactor by an Aspergillus foetidus strain isolated from the Brazilian savanna soil using different nitrogen sources. Its maximum activity (63.7 U mL -1 ) was obtained in a medium containing 2% (w/v) peptone. A cultivation carried out in a 5.0 L stirred-tank bioreactor provided a maximum protease activity 9% lower than that observed in Erlenmeyer flasks, which was obtained after a significantly shorter (by 16-29%) time. Protease purification by a combination of gel-filtration chromatography resulted in a 16.9-fold increase in specific activity (248.1 U g -1 ). The estimated molecular weight of the purified enzyme was 50.6 kDa, and the optimal pH and temperature were 5.0 and 55 °C, respectively. The enzyme was completely inhibited by pepstatin A, and its activity enhanced by some metals. According to the inhibition profiles, it was confirmed that the purified acid protease belongs to the aspartic protease type. These results are quite promising for future development of large-scale production of such protease, which can be useful in biotechnological applications requiring high enzyme activity and stability under acidic conditions. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Expression and characterization of plant aspartic protease nepenthesin-1 from Nepenthes gracilis.

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

    2014-03-01

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

  16. Molecular cloning and immunochemical characterization of a novel major Japanese cedar pollen allergen belonging to the aspartic protease family.

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

    2010-01-01

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

  17. CTAB Zymography for the Analysis of Aspartic Proteases from Marine Sponges.

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    González, Oscar; Wilkesman, Jeff

    2017-01-01

    Electrophoresis under denaturing conditions in the presence of SDS is a standard method for the protein and enzyme scientist. Nevertheless, there are special situations where this method may originate nonoptimal results. SDS may cause protein aggregation or precipitation. Beyond this, depending on the type of protein, some just do not resolve well or migrate abnormally in SDS gels. SDS, an anionic detergent, may be however substituted by a cationic detergent, like CTAB (cetyltrimethylammonium bromide), in order to solubilize and electrophorize proteins. CTAB electrophoresis allows the separation of proteins based on molecular weight and can be carried out at neutral or acidic pH. Here, we describe the development of a CTAB zymography method to analyze aspartic proteases from marine sponges, which present an abnormal high R f value when run in SDS-PAGE. The special feature of using CTAB is that it binds proteins, making them positively charged and thus migrating in the opposite direction compared to SDS-PAGE.

  18. Potential antioxidant peptides produced from whey hydrolysis with an immobilized aspartic protease from Salpichroa origanifolia fruits.

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    Rocha, Gabriela Fernanda; Kise, Francisco; Rosso, Adriana Mabel; Parisi, Mónica Graciela

    2017-12-15

    An aspartic protease from Salpichroa origanifolia fruits was successfully immobilized onto an activated support of glutaraldehyde agarose. The immobilized enzyme presented higher thermal stability than the free enzyme from 40°C to 50°C and high reusability, retaining 54% of the initial activity after ten cycles of the process. Whey protein concentrates (WPC) were hydrolyzed with both free and immobilized enzyme, reaching a similar degree of hydrolysis of approximately 6-8% after 20h. In addition, the immobilized derivate hydrolyzed α-lactalbumin protein with a higher affinity than β-lactoglobulin. The hydrolysate was ultra-filtrated, and the fractions were evaluated for antioxidant activities with the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity method. The fraction containing peptides with a molecular mass below 3kDa demonstrated a strong radical quenching effect (IC 50: 0.48mg/ml). These results suggest that hydrolyzed WPC could be considered as a promising source of natural food antioxidants for the development of functional food. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. A novel aspartic acid protease gene from pineapple fruit (Ananas comosus): cloning, characterization and relation to postharvest chilling stress resistance.

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    Raimbault, Astrid-Kim; Zuily-Fodil, Yasmine; Soler, Alain; Cruz de Carvalho, Maria H

    2013-11-15

    A full-length cDNA encoding a putative aspartic acid protease (AcAP1) was isolated for the first time from the flesh of pineapple (Ananas comosus) fruit. The deduced sequence of AcAP1 showed all the common features of a typical plant aspartic protease phytepsin precursor. Analysis of AcAP1 gene expression under postharvest chilling treatment in two pineapple varieties differing in their resistance to blackheart development revealed opposite trends. The resistant variety showed an up-regulation of AcAP1 precursor gene expression whereas the susceptible showed a down-regulation in response to postharvest chilling treatment. The same trend was observed regarding specific AP enzyme activity in both varieties. Taken together our results support the involvement of AcAP1 in postharvest chilling stress resistance in pineapple fruits. Copyright © 2013 Elsevier GmbH. All rights reserved.

  20. Aspartic protease inhibitory and nematocidal activity of phenyl-4-(2-phenylhydrazonohexahydrofuro[3,2-c]pyridazin-7-ol (Percival dianhydroosazone

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    El Sayed H. El Ashry

    2014-04-01

    Full Text Available We synthesized Phenyl-4-(2-phenylhydrazonohexahydrofuro[3,2-c]pyridazin-7-ol (compound 3. The structure compound 3 was elucidated with IR, 1H NMR, 13C NMR and EIMS spectra. Compound 3 showed potent inhibitory activity against aspartic proteases, human cathepsin D and Plasmodium falciparum plasmepsin-II with IC50 = 20 μM. Enzyme-inhibitor complexes were predicted to stabilize by electrostatic and hydrophobic interactions between the side chains of amino acid residues at the active center and compound 3. Moreover, compound 3 displayed good nematocidal activity against all developmental stages of C. elegans.

  1. Secreted aspartic proteases of pathogenic Candida spp. are temporarily retained in the cell wall and cleave the extracellular substrates

    Czech Academy of Sciences Publication Activity Database

    Pichová, Iva; Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga

    2012-01-01

    Roč. 21, S1 (2012), s. 206-206 ISSN 0961-8368. [Annual Symposium of the Protein-Society /26./. 05.08.2012-08.08.2012, San Diego] R&D Projects: GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : aspartic proteases * Candida spp. * cell wall Subject RIV: CE - Biochemistry

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

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

    2010-09-01

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

  3. Genome-wide identification, evolutionary and expression analysis of the aspartic protease gene superfamily in grape

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    2013-01-01

    Background Aspartic proteases (APs) are a large family of proteolytic enzymes found in almost all organisms. In plants, they are involved in many biological processes, such as senescence, stress responses, programmed cell death, and reproduction. Prior to the present study, no grape AP gene(s) had been reported, and their research on woody species was very limited. Results In this study, a total of 50 AP genes (VvAP) were identified in the grape genome, among which 30 contained the complete ASP domain. Synteny analysis within grape indicated that segmental and tandem duplication events contributed to the expansion of the grape AP family. Additional analysis between grape and Arabidopsis demonstrated that several grape AP genes were found in the corresponding syntenic blocks of Arabidopsis, suggesting that these genes arose before the divergence of grape and Arabidopsis. Phylogenetic relationships of the 30 VvAPs with the complete ASP domain and their Arabidopsis orthologs, as well as their gene and protein features were analyzed and their cellular localization was predicted. Moreover, expression profiles of VvAP genes in six different tissues were determined, and their transcript abundance under various stresses and hormone treatments were measured. Twenty-seven VvAP genes were expressed in at least one of the six tissues examined; nineteen VvAPs responded to at least one abiotic stress, 12 VvAPs responded to powdery mildew infection, and most of the VvAPs responded to SA and ABA treatments. Furthermore, integrated synteny and phylogenetic analysis identified orthologous AP genes between grape and Arabidopsis, providing a unique starting point for investigating the function of grape AP genes. Conclusions The genome-wide identification, evolutionary and expression analyses of grape AP genes provide a framework for future analysis of AP genes in defining their roles during stress response. Integrated synteny and phylogenetic analyses provide novel insight into the

  4. Aspartic protease from Aspergillus (Eurotium) repens strain MK82 is involved in the hydrolysis and decolourisation of dried bonito (Katsuobushi).

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    Aoki, Kenji; Matsubara, Sayaka; Umeda, Mayo; Tachibanac, Shusaku; Doi, Mikiharu; Takenaka, Shinji

    2013-04-01

    Katsuobushi is a dried, smoked and fermented bonito used in Japanese cuisine. During the fermentation process with several Aspergillus species, the colour of Katsuobushi gradually changes from a dark reddish-brown derived from haem proteins to pale pink. The change in colour gives Katsuobushi a higher ranking and price. This study aimed to elucidate the mechanism of decolourisation of Katsuobushi. A decolourising factor from the culture supernatant of Aspergillus (Eurotium) repens strain MK82 was purified to homogeneity. The purification was monitored by measuring the decolourising activity using equine myoglobin and bovine haemoglobin as substrates. It was found that the decolourising factor had protease activity towards myoglobin and haemoglobin. Complete inhibition of the enzyme by the inhibitor pepstatin A and the internal amino acid sequence classified the protein as an aspartic protease. The enzyme limitedly hydrolysed myoglobin between 1-Met and 2-Gly, 43-Lys and 44-Phe, and 70-Leu and 71-Thr. The purified enzyme decolourised blood of Katsuwonus pelamis (bonito) and a slice of dried bonito. It is proposed that aspartic protease plays a role in the decolourisation of Katsuobushi by the hydrolysis of haem proteins that allows the released haem to aggregate in the dried bonito. © 2012 Society of Chemical Industry.

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

    Science.gov (United States)

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

    2017-01-01

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

  6. Inhibition of xyloglucanase from an alkalothermophilic Thermomonospora sp. by a peptidic aspartic protease inhibitor from Penicillium sp. VM24.

    Science.gov (United States)

    Menon, Vishnu; Rao, Mala

    2012-11-01

    A bifunctional inhibitor from Penicillium sp VM24 causing inactivation of xyloglucanase from Thermomonospora sp and an aspartic protease from Aspergillus saitoi was identified. Steady state kinetics studies of xyloglucanase and the inhibitor revealed an irreversible, non-competitive, two-step inhibition mechanism with IC(50) and K(i) values of 780 and 500nM respectively. The interaction of o-phthalaldehyde (OPTA)-labeled xyloglucanase with the inhibitor revealed that the inhibitor binds to the active site of the enzyme. Far- and near-UV spectrophotometric analysis suggests that the conformational changes induced in xyloglucanase by the inhibitor may be due to irreversible denaturation of enzyme. The bifunctional inhibitor may have potential as a biocontrol agent for the protection of plants against phytopathogenic fungi. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Comparative structure-function characterization of the saposin-like domains from potato, barley, cardoon and Arabidopsis aspartic proteases.

    Science.gov (United States)

    Bryksa, Brian C; Grahame, Douglas A; Yada, Rickey Y

    2017-05-01

    The present study characterized the aspartic protease saposin-like domains of four plant species, Solanum tuberosum (potato), Hordeum vulgare L. (barley), Cynara cardunculus L. (cardoon; artichoke thistle) and Arabidopsis thaliana, in terms of bilayer disruption and fusion, and structure pH-dependence. Comparison of the recombinant saposin-like domains revealed that each induced leakage of bilayer vesicles composed of a simple phospholipid mixture with relative rates Arabidopsis>barley>cardoon>potato. When compared for leakage of bilayer composed of a vacuole-like phospholipid mixture, leakage was approximately five times higher for potato saposin-like domain compared to the others. In terms of fusogenic activity, distinctions between particle size profiles were noted among the four proteins, particularly for potato saposin-like domain. Bilayer fusion assays in reducing conditions resulted in altered fusion profiles except in the case of cardoon saposin-like domain which was virtually unchanged. Secondary structure profiles were similar across all four proteins under different pH conditions, although cardoon saposin-like domain appeared to have higher overall helix structure. Furthermore, increases in Trp emission upon protein-bilayer interactions suggested that protein structure rearrangements equilibrated with half-times ranging from 52 to 120s, with cardoon saposin-like domain significantly slower than the other three species. Overall, the present findings serve as a foundation for future studies seeking to delineate protein structural features and motifs in protein-bilayer interactions based upon variability in plant aspartic protease saposin-like domain structures. Copyright © 2017 Elsevier B.V. All rights reserved.

  8. Monitoring the impact of an aspartic protease (MpAPr1) on grape proteins and wine properties.

    Science.gov (United States)

    Theron, Louwrens Wiid; Bely, Marina; Divol, Benoit

    2018-04-23

    The perception of haze in wine is brought about when pathogenesis-related proteins become unstable and aggregate, subsequently resulting in crosslinking until it develops into light-dispersing particles. Elimination of these proteins is usually achieved via bentonite fining, which, although effective, suffers from several drawbacks. The utilization of proteases has been proposed as an ideal alternative. In a previous study, an aspartic protease (MpAPr1) from the yeast Metschnikowia pulcherrima was purified and shown to be partially active against grape proteins in synthetic medium. In this study, the effects of pure MpAPr1 supplemented to Sauvignon Blanc juice on subsequent fermentation were investigated. The juice was incubated for 48 h and thereafter inoculated with Saccharomyces cerevisiae. Results revealed that the enzyme had no observable effects on fermentation performance and retained activity throughout. Protein degradation could be detected and resulted in a significant modification of the wine composition and an increase in the presence of certain volatile compounds, especially those linked to amino acid metabolism.

  9. A Novel Aspartic Protease with HIV-1 Reverse Transcriptase Inhibitory Activity from Fresh Fruiting Bodies of the Wild Mushroom Xylaria hypoxylon

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    Qing-Xiu Hu

    2012-01-01

    Full Text Available A novel aspartic protease with HIV-1 RT inhibitory activity was isolated and characterized from fruiting bodies of the wild mushroom Xylaria hypoxylon. The purification protocol comprised distilled water homogenization and extraction step, three ion exchange chromatographic steps (on DEAE-cellulose, Q-Sepharose, and CM-cellulose in succession, and final purification was by FPLC on Superdex 75. The protease was adsorbed on all the three ion exchangers. It was a monomeric protein with a molecular mass of 43 kDa as estimated by SDS-PAGE and FPLC. Its N-terminal amino acid sequence was HYTELLSQVV, which exhibited no sequence homology to other proteases reported. The activity of the protease was adversely affected by Pepstatin A, indicating that it is an aspartic protease. The protease activity was maximal or nearly so in the pH range 6–8 and in the temperature range 35–60°C. The purified enzyme exhibited HIV-1 RT inhibitory activity with an IC50 value of 8.3 μM, but was devoid of antifungal, ribonuclease, and hemagglutinating activities.

  10. Age estimation based on aspartic acid racemization in human sclera.

    Science.gov (United States)

    Klumb, Karolin; Matzenauer, Christian; Reckert, Alexandra; Lehmann, Klaus; Ritz-Timme, Stefanie

    2016-01-01

    Age estimation based on racemization of aspartic acid residues (AAR) in permanent proteins has been established in forensic medicine for years. While dentine is the tissue of choice for this molecular method of age estimation, teeth are not always available which leads to the need to identify other suitable tissues. We examined the suitability of total tissue samples of human sclera for the estimation of age at death. Sixty-five samples of scleral tissue were analyzed. The samples were hydrolyzed and after derivatization, the extent of aspartic acid racemization was determined by gas chromatography. The degree of AAR increased with age. In samples from younger individuals, the correlation of age and D-aspartic acid content was closer than in samples from older individuals. The age-dependent racemization in total tissue samples proves that permanent or at least long-living proteins are present in scleral tissue. The correlation of AAR in human sclera and age at death is close enough to serve as basis for age estimation. However, the precision of age estimation by this method is lower than that of age estimation based on the analysis of dentine which is due to molecular inhomogeneities of total tissue samples of sclera. Nevertheless, the approach may serve as a valuable alternative or addition in exceptional cases.

  11. Enzymatic milk clotting activity in artichoke (Cynara scolymus) leaves and alpine thistle (Carduus defloratus) flowers. Immobilization of alpine thistle aspartic protease.

    Science.gov (United States)

    Esposito, Marilena; Di Pierro, Prospero; Dejonghe, Winnie; Mariniello, Loredana; Porta, Raffaele

    2016-08-01

    Two different milk clotting enzymes, belonging to the aspartic protease family, were extracted from both artichoke leaves and alpine thistle flowers, and the latter was covalently immobilized by using a polyacrylic support containing polar epoxy groups. Our findings showed that the alpine thistle aspartic protease was successfully immobilized at pH 7.0 on Immobeads IB-150P beads and that, under these experimental conditions, an immobilization yield of about 68% and a recovery of about 54% were obtained. Since the enzyme showed an optimal pH of 5.0, a value very similar to the one generally used for milk clotting during cheese making, and exhibited a satisfactory stability over time, the use of such immobilized vegetable rennet for the production of novel dairy products is suggested. Copyright © 2016. Published by Elsevier Ltd.

  12. Secreted Aspartic Protease Cleavage of Candida albicans Msb2 Activates Cek1 MAPK Signaling Affecting Biofilm Formation and Oropharyngeal Candidiasis

    Science.gov (United States)

    Chadha, Sonia; Tati, Swetha; Conti, Heather R.; Hube, Bernhard; Cullen, Paul J.; Edgerton, Mira

    2012-01-01

    Perception of external stimuli and generation of an appropriate response are crucial for host colonization by pathogens. In pathogenic fungi, mitogen activated protein kinase (MAPK) pathways regulate dimorphism, biofilm/mat formation, and virulence. Signaling mucins, characterized by a heavily glycosylated extracellular domain, a transmembrane domain, and a small cytoplasmic domain, are known to regulate various signaling pathways. In Candida albicans, the mucin Msb2 regulates the Cek1 MAPK pathway. We show here that Msb2 is localized to the yeast cell wall and is further enriched on hyphal surfaces. A msb2Δ/Δ strain formed normal hyphae but had biofilm defects. Cek1 (but not Mkc1) phosphorylation was absent in the msb2Δ/Δ mutant. The extracellular domain of Msb2 was shed in cells exposed to elevated temperature and carbon source limitation, concomitant with germination and Cek1 phosphorylation. Msb2 shedding occurred differentially in cells grown planktonically or on solid surfaces in the presence of cell wall and osmotic stressors. We further show that Msb2 shedding and Cek1 phosphorylation were inhibited by addition of Pepstatin A (PA), a selective inhibitor of aspartic proteases (Saps). Analysis of combinations of Sap protease mutants identified a sap8Δ/Δ mutant with reduced MAPK signaling along with defects in biofilm formation, thereby suggesting that Sap8 potentially serves as a major regulator of Msb2 processing. We further show that loss of either Msb2 (msb2Δ/Δ) or Sap8 (sap8Δ/Δ) resulted in higher C. albicans surface β-glucan exposure and msb2Δ/Δ showed attenuated virulence in a murine model of oral candidiasis. Thus, Sap-mediated proteolytic cleavage of Msb2 is required for activation of the Cek1 MAPK pathway in response to environmental cues including those that induce germination. Inhibition of Msb2 processing at the level of Saps may provide a means of attenuating MAPK signaling and reducing C. albicans virulence. PMID:23139737

  13. Identification of Novel Placentally Expressed Aspartic Proteinase in Humans

    Directory of Open Access Journals (Sweden)

    Marta Majewska

    2017-06-01

    Full Text Available This study presents pioneering data concerning the human pregnancy-associated glycoprotein-Like family, identified in the genome, of the term placental transcriptome and proteome. RNA-seq allowed the identification of 1364 bp hPAG-L/pep cDNA with at least 56.5% homology with other aspartic proteinases (APs. In silico analyses revealed 388 amino acids (aa of full-length hPAG-L polypeptide precursor, with 15 aa-signal peptide, 47 aa-blocking peptide and 326 aa-mature protein, and two Asp residues (D, specific for a catalytic cleft of the APs (VVFDTGSSNLWV91-102 and AIVDTGTSLLTG274-285. Capillary sequencing identified 9330 bp of the hPAG-L gene (Gen Bank Acc. No. KX533473, composed of nine exons and eight introns. Heterologous Western blotting revealed the presence of one dominant 60 kDa isoform of the hPAG-L amongst cellular placental proteins. Detection with anti-pPAG-P and anti-Rec pPAG2 polyclonals allowed identification of the hPAG-L proteins located within regions of chorionic villi, especially within the syncytiotrophoblast of term singleton placentas. Our novel data extend the present knowledge about the human genome, as well as placental transcriptome and proteome during term pregnancy. Presumably, this may contribute to establishing a new diagnostic tool for examination of some disturbances during human pregnancy, as well as growing interest from both scientific and clinical perspectives.

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

    Directory of Open Access Journals (Sweden)

    Madhusudhan Budatha

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

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

    Science.gov (United States)

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

    2017-07-01

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

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

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    Chen Chaoping

    2010-07-01

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

  17. 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)

  18. In silico insights into protein-protein interactions and folding dynamics of the saposin-like domain of Solanum tuberosum aspartic protease.

    Directory of Open Access Journals (Sweden)

    Dref C De Moura

    Full Text Available The plant-specific insert is an approximately 100-residue domain found exclusively within the C-terminal lobe of some plant aspartic proteases. Structurally, this domain is a member of the saposin-like protein family, and is involved in plant pathogen defense as well as vacuolar targeting of the parent protease molecule. Similar to other members of the saposin-like protein family, most notably saposins A and C, the recently resolved crystal structure of potato (Solanum tuberosum plant-specific insert has been shown to exist in a substrate-bound open conformation in which the plant-specific insert oligomerizes to form homodimers. In addition to the open structure, a closed conformation also exists having the classic saposin fold of the saposin-like protein family as observed in the crystal structure of barley (Hordeum vulgare L. plant-specific insert. In the present study, the mechanisms of tertiary and quaternary conformation changes of potato plant-specific insert were investigated in silico as a function of pH. Umbrella sampling and determination of the free energy change of dissociation of the plant-specific insert homodimer revealed that increasing the pH of the system to near physiological levels reduced the free energy barrier to dissociation. Furthermore, principal component analysis was used to characterize conformational changes at both acidic and neutral pH. The results indicated that the plant-specific insert may adopt a tertiary structure similar to the characteristic saposin fold and suggest a potential new structural motif among saposin-like proteins. To our knowledge, this acidified PSI structure presents the first example of an alternative saposin-fold motif for any member of the large and diverse SAPLIP family.

  19. Cold-adapted digestive aspartic protease of the clawed lobsters Homarus americanus and Homarus gammarus: biochemical characterization.

    Science.gov (United States)

    Rojo, Liliana; García-Carreño, Fernando; de Los Angeles Navarrete del Toro, Maria

    2013-02-01

    Aspartic proteinases in the gastric fluid of clawed lobsters Homarus americanus and Homarus gammarus were isolated to homogeneity by single-step pepstatin-A affinity chromatography; such enzymes have been previously identified as cathepsin D-like enzymes based on their deduced amino acid sequence. Here, we describe their biochemical characteristics; the properties of the lobster enzymes were compared with those of its homolog, bovine cathepsin D, and found to be unique in a number of ways. The lobster enzymes demonstrated hydrolytic activity against synthetic and natural substrates at a wider range of pH; they were more temperature-sensitive, showed no changes in the K(M) value at 4°C, 10°C, and 25°C, and had 20-fold higher k(cat)/K(M) values than bovine enzyme. The bovine enzyme was temperature-dependent. We propose that both properties arose from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low habitat temperatures. This is supported by the fast denaturation rates induced by temperature.

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

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    Surasak Jittavisutthikul

    2015-04-01

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

  1. Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment-Based Drug Design Facilitated by Dynamic Combinatorial Chemistry.

    Science.gov (United States)

    Mondal, Milon; Radeva, Nedyalka; Fanlo-Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard; Hirsch, Anna K H

    2016-08-01

    Fragment-based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit-identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X-ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis-acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240-fold improvement in potency compared to the parent hits. Subsequent X-ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit-identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit-to-lead optimization. © 2016 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  2. Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

    Science.gov (United States)

    Mondal, Milon; Radeva, Nedyalka; Fanlo‐Virgós, Hugo; Otto, Sijbren; Klebe, Gerhard

    2016-01-01

    Abstract Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC50 value of 54 nm, which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization. PMID:27400756

  3. Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid

    NARCIS (Netherlands)

    Sivan, S.S.; Tsitron, E.; Wachtel, E.; Roughley, P.J.; Sakkee, N.; Ham, F. van der; Groot, J.de; Roberts, S.; Maroudas, A.

    2006-01-01

    We have used the racemization of aspartic acid as a marker for the "molecular age" of aggrecan components of the human intervertebral disc matrix (aggregating and non-aggregating proteoglycans as well as the different buoyant density fractions of aggrecan). By measuring the D/L Asp ratio of the

  4. Aspartic acid

    Science.gov (United States)

    ... we eat. Aspartic acid is also called asparaginic acid. Aspartic acid helps every cell in the body work. It ... release Normal nervous system function Plant sources of aspartic acid include: avocado, asparagus, and molasses. Animal sources of ...

  5. Do alterations in follicular fluid proteases contribute to human infertility?

    Science.gov (United States)

    Cookingham, Lisa Marii; Van Voorhis, Bradley J; Ascoli, Mario

    2015-05-01

    Cathepsin L and ADAMTS-1 are known to play critical roles in follicular rupture, ovulation, and fertility in mice. Similar studies in humans are limited; however, both are known to increase during the periovulatory period. No studies have examined either protease in the follicular fluid of women with unexplained infertility or infertility related to advanced maternal age (AMA). We sought to determine if alterations in cathepsin L and/or ADAMTS-1 existed in these infertile populations. Patients undergoing in vitro fertilization (IVF) for unexplained infertility or AMA-related infertility were prospectively recruited for the study; patients with tubal or male factor infertility were recruited as controls. Follicular fluid was collected to determine gene expression (via quantitative polymerase chain reaction), enzyme concentrations (via enzyme-linked immunosorbent assays), and enzymatic activities (via fluorogenic enzyme cleavage assay or Western blot analysis) of cathepsin L and ADAMTS-1. The analysis included a total of 42 patients (14 per group). We found no statistically significant difference in gene expression, enzyme concentration, or enzymatic activity of cathepsin L or ADAMTS-1 in unexplained infertility or AMA-related infertility as compared to controls. We also found no statistically significant difference in expression or concentration with advancing age. Cathepsin L and ADAMTS-1 are not altered in women with unexplained infertility or AMA-related infertility undergoing IVF, and they do not decline with advancing age. It is possible that differences exist in natural cycles, contributing to infertility; however, our findings do not support a role for protease alterations as a common cause of infertility.

  6. Protease activation involved in resistance of human cells to x-ray cell killing

    International Nuclear Information System (INIS)

    Zhang, Hong-Chang; Takahashi, Shuji; Karata, Kiyonobu; Kita, Kazuko; Suzuki, Nobuo

    2003-01-01

    Little is known of proteases that play roles in the early steps of X-ray irradiation response. In the present study, we first searched for proteases whose activity is induced in human RSa-R cells after X-ray irradiation. The activity was identified as fibrinolytic, using 125 I-labeled fibrin as a substrate. Protease samples were prepared by lysation of cells with a buffer containing MEGA-8. RSa-R cells showed an increased level of protease activity 10 min after X-ray (up to 3 Gy) irradiation. We next examined whether this protease inducibility is causally related with the X-ray susceptibility of cells. Leupeptin, a serine-cysteine protease inhibitor, inhibited the protease activity in samples obtained from X-ray-irradiated RSa-R cells. Treatment of RSa-R cells with the inhibitor before and after X-ray irradiation resulted in an increased susceptibility of the cells to X-ray cell killing. However, the treatment of cells with other inhibitors tested did not modulate the X-ray susceptibility. These results suggest that leupeptin-sensitive proteases are involved in the resistance of human cells to X-ray cell killing. (author)

  7. Cloning and expression of the human N-methyl-D-aspartate receptor subunit NR3A

    DEFF Research Database (Denmark)

    Eriksson, Maria; Nilsson, Anna; Froelich-Fabre, Susanne

    2002-01-01

    Native N-methyl-D-aspartate (NMDA) receptors are heteromeric assemblies of four or five subunits. The NMDA receptor subunits, NR1, NR2A, NR2B, NR2C, and NR2D have been cloned in several species, including man. The NR3A subunit, which in rodents is predominantly expressed during early development......, seems to function by reducing the NMDA receptor response. The human homologue to the rat NR3A, however, had not been cloned. In order to study the functions of the human NR3A (hNR3A), we have cloned and sequenced the hNR3A. It was found to share 88% of the DNA sequence with the rat gene, corresponding...

  8. Exposure to ozone modulates human airway protease/antiprotease balance contributing to increased influenza A infection.

    Directory of Open Access Journals (Sweden)

    Matthew J Kesic

    Full Text Available Exposure to oxidant air pollution is associated with increased respiratory morbidities and susceptibility to infections. Ozone is a commonly encountered oxidant air pollutant, yet its effects on influenza infections in humans are not known. The greater Mexico City area was the primary site for the spring 2009 influenza A H1N1 pandemic, which also coincided with high levels of environmental ozone. Proteolytic cleavage of the viral membrane protein hemagglutinin (HA is essential for influenza virus infectivity. Recent studies suggest that HA cleavage might be cell-associated and facilitated by the type II transmembrane serine proteases (TTSPs human airway trypsin-like protease (HAT and transmembrane protease, serine 2 (TMPRSS2, whose activities are regulated by antiproteases, such as secretory leukocyte protease inhibitor (SLPI. Based on these observations, we sought to determine how acute exposure to ozone may modulate cellular protease/antiprotease expression and function, and to define their roles in a viral infection. We utilized our in vitro model of differentiated human nasal epithelial cells (NECs to determine the effects of ozone on influenza cleavage, entry, and replication. We show that ozone exposure disrupts the protease/antiprotease balance within the airway liquid. We also determined that functional forms of HAT, TMPRSS2, and SLPI are secreted from human airway epithelium, and acute exposure to ozone inversely alters their expression levels. We also show that addition of antioxidants significantly reduces virus replication through the induction of SLPI. In addition, we determined that ozone-induced cleavage of the viral HA protein is not cell-associated and that secreted endogenous proteases are sufficient to activate HA leading to a significant increase in viral replication. Our data indicate that pre-exposure to ozone disrupts the protease/antiprotease balance found in the human airway, leading to increased influenza susceptibility.

  9. Counter-regulatory hormone responses to spontaneous hypoglycaemia during treatment with insulin Aspart or human soluble insulin

    DEFF Research Database (Denmark)

    Brock Jacobsen, I; Vind, B F; Korsholm, Lars

    2011-01-01

    examined in a randomized, double-blinded cross-over study for two periods of 8 weeks. Sixteen patients with type 1 diabetes were subjected to three daily injections of human soluble insulin or Aspart in addition to Neutral Protamine Hagedorn (NPH) insulin twice daily. Each intervention period was followed......-regulatory responses regarding growth hormone, glucagon and ghrelin whereas no differences were found in relation to free fatty acid, cortisol, insulin-like growth factor (IGF)-I, IGF-II and IGF-binding proteins 1 and 2. Treatment with insulin Aspart resulted in well-defined peaks in serum insulin concentrations...... elicited a slightly different physiological response to spontaneous hypoglycaemia compared with human insulin. Keywords hypoglycaemia counter-regulation, insulin Aspart, type 1 diabetes....

  10. Retroviral proteases and their roles in virion maturation

    Czech Academy of Sciences Publication Activity Database

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

    2015-01-01

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

  11. Collagen turnover in normal and degenerate human intervertebral discs as determined by the racemization of aspartic acid

    NARCIS (Netherlands)

    Sivan, S.-S.; Wachtel, E.; Tsitron, E.; Sakkee, N.; Ham, F. van der; Groot, J.de; Roberts, S.; Maroudas, A.

    2008-01-01

    Knowledge of rates of protein turnover is important for a quantitative understanding of tissue synthesis and catabolism. In this work, we have used the racemization of aspartic acid as a marker for the turnover of collagen obtained from healthy and pathological human intervertebral disc matrices. We

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

    Science.gov (United States)

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

    1992-08-01

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

  13. Possible identity of IL-8 converting enzyme in human fibroblasts as a cysteine protease.

    Science.gov (United States)

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

    2003-04-01

    A converting activity was characterized in human diploid fibroblasts, which secrete 72IL-8 and 77IL-8 in treatment with IFN-beta and poly I: poly C. 77IL-8 was significantly converted to 72IL-8 by a partially purified fraction of the culture supernatant of human diploid fibroblasts. The converting activity, which was temperature-dependent and optimal at pH 6, was completely inhibited by cysteine protease inhibitors, antipain dihydrochloride and E-64, but not by other types of protease inhibitors. These data clearly show that human diploid fibroblasts are capable of processing IL-8 to produce a mature IL-8 and that the putative converting enzyme appears to be a cysteine protease.

  14. Human mast cell neutral proteases generate modified LDL particles with increased proteoglycan binding.

    Science.gov (United States)

    Maaninka, Katariina; Nguyen, Su Duy; Mäyränpää, Mikko I; Plihtari, Riia; Rajamäki, Kristiina; Lindsberg, Perttu J; Kovanen, Petri T; Öörni, Katariina

    2018-04-13

    Subendothelial interaction of LDL with extracellular matrix drives atherogenesis. This interaction can be strengthened by proteolytic modification of LDL. Mast cells (MCs) are present in atherosclerotic lesions, and upon activation, they degranulate and release a variety of neutral proteases. Here we studied the ability of MC proteases to cleave apoB-100 of LDL and affect the binding of LDL to proteoglycans. Mature human MCs were differentiated from human peripheral blood-derived CD34 + progenitors in vitro and activated with calcium ionophore to generate MC-conditioned medium. LDL was incubated in the MC-conditioned medium or with individual MC proteases, and the binding of native and modified LDL to isolated human aortic proteoglycans or to human atherosclerotic plaques ex vivo was determined. MC proteases in atherosclerotic human coronary artery lesions were detected by immunofluorescence and qPCR. Activated human MCs released the neutral proteases tryptase, chymase, carboxypeptidase A3, cathepsin G, and granzyme B. Of these, cathepsin G degraded most efficiently apoB-100, induced LDL fusion, and enhanced binding of LDL to isolated human aortic proteoglycans and human atherosclerotic lesions ex vivo. Double immunofluoresence staining of human atherosclerotic coronary arteries for tryptase and cathepsin G indicated that lesional MCs contain cathepsin G. In the lesions, expression of cathepsin G correlated with the expression of tryptase and chymase, but not with that of neutrophil proteinase 3. The present study suggests that cathepsin G in human atherosclerotic lesions is largely derived from MCs and that activated MCs may contribute to atherogenesis by enhancing LDL retention. Copyright © 2018 Elsevier B.V. All rights reserved.

  15. Substitution of aspartic acid-686 by histidine or asparagine in the human androgen receptor leads to a functionally inactive protein with altered hormone-binding characteristics

    NARCIS (Netherlands)

    Ris-Stalpers, C.; Trifiro, M. A.; Kuiper, G. G.; Jenster, G.; Romalo, G.; Sai, T.; van Rooij, H. C.; Kaufman, M.; Rosenfield, R. L.; Liao, S.

    1991-01-01

    We have identified two different single nucleotide alterations in codon 686 (GAC; aspartic acid) in exon 4 of the human androgen receptor gene in three unrelated families with the complete form of androgen insensitivity. One mutation (G----C) results in an aspartic acid----histidine substitution

  16. Human cerebrospinal fluid monoclonal N-methyl-D-aspartate receptor autoantibodies are sufficient for encephalitis pathogenesis.

    Science.gov (United States)

    Kreye, Jakob; Wenke, Nina K; Chayka, Mariya; Leubner, Jonas; Murugan, Rajagopal; Maier, Nikolaus; Jurek, Betty; Ly, Lam-Thanh; Brandl, Doreen; Rost, Benjamin R; Stumpf, Alexander; Schulz, Paulina; Radbruch, Helena; Hauser, Anja E; Pache, Florence; Meisel, Andreas; Harms, Lutz; Paul, Friedemann; Dirnagl, Ulrich; Garner, Craig; Schmitz, Dietmar; Wardemann, Hedda; Prüss, Harald

    2016-10-01

    SEE ZEKERIDOU AND LENNON DOI101093/AWW213 FOR A SCIENTIFIC COMMENTARY ON THIS ARTICLE: Anti-N-methyl-D-aspartate receptor (NMDAR) encephalitis is a recently discovered autoimmune syndrome associated with psychosis, dyskinesias, and seizures. Little is known about the cerebrospinal fluid autoantibody repertoire. Antibodies against the NR1 subunit of the NMDAR are thought to be pathogenic; however, direct proof is lacking as previous experiments could not distinguish the contribution of further anti-neuronal antibodies. Using single cell cloning of full-length immunoglobulin heavy and light chain genes, we generated a panel of recombinant monoclonal NR1 antibodies from cerebrospinal fluid memory B cells and antibody secreting cells of NMDAR encephalitis patients. Cells typically carried somatically mutated immunoglobulin genes and had undergone class-switching to immunoglobulin G, clonally expanded cells carried identical somatic hypermutation patterns. A fraction of NR1 antibodies were non-mutated, thus resembling 'naturally occurring antibodies' and indicating that tolerance induction against NMDAR was incomplete and somatic hypermutation not essential for functional antibodies. However, only a small percentage of cerebrospinal fluid-derived antibodies reacted against NR1. Instead, nearly all further antibodies bound specifically to diverse brain-expressed epitopes including neuronal surfaces, suggesting that a broad repertoire of antibody-secreting cells enrich in the central nervous system during encephalitis. Our functional data using primary hippocampal neurons indicate that human cerebrospinal fluid-derived monoclonal NR1 antibodies alone are sufficient to cause neuronal surface receptor downregulation and subsequent impairment of NMDAR-mediated currents, thus providing ultimate proof of antibody pathogenicity. The observed formation of immunological memory might be relevant for clinical relapses. © The Author (2016). Published by Oxford University Press on

  17. Ninety-nine is not enough: molecular characterization of inhibitor-resistant human immunodeficiency virus type 1 protease mutants with insertions in the flap region

    Czech Academy of Sciences Publication Activity Database

    Kožíšek, Milan; Grantz Šašková, Klára; Řezáčová, Pavlína; Brynda, Jiří; Maarseveen van, N. M.; De Jongh, D.; Boucher, Ch. A. B.; Kagan, R. M.; Nijhuis, M.; Konvalinka, Jan

    2008-01-01

    Roč. 82, č. 12 (2008), s. 5869-5878 ISSN 0022-538X R&D Projects: GA MŠk 1M0508; GA MZd NR8571 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : HIV protease inhibitors * aspartic proteases * viral resistance * insertions Subject RIV: CE - Biochemistry Impact factor: 5.308, year: 2008

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

    Directory of Open Access Journals (Sweden)

    Raheem Ullah

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

  19. 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.

  20. Use of artichoke (Cynara scolymus) flower extract as a substitute for bovine rennet in the manufacture of Gouda-type cheese: characterization of aspartic proteases.

    Science.gov (United States)

    Llorente, Berta E; Obregón, Walter David; Avilés, Francesc X; Caffini, Néstor O; Vairo-Cavalli, Sandra

    2014-09-15

    Artichoke (Cynara scolymus L.) flower extract was assayed with the aim of replacing animal rennet in the manufacture of Gouda-type cheeses from bovine milk. Floral extract coagulated milk within a suitable time for use on an industrial scale, while the yield of cheese obtained was equal to that achieved with bovine abomasum. Five proteolytic fractions with milk-clotting activity were isolated in a two-step purification protocol, three belonging to the cardosin group. Cheeses made with C. scolymus proteases must be brined for a longer period (40 h) to prevent overproteolysis and avoid the development of a background flavor. The type of coagulant (bovine or vegetable) had no significant effect on the cheeses' chemical parameters analyzed throughout ripening, and no significant organoleptic differences were detected between those manufactured with C. scolymus or animal rennet. The results indicate that C. scolymus flower extract is suitable for replacing animal rennet in the production of Gouda-type cheeses. Copyright © 2014 Elsevier Ltd. All rights reserved.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    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...

  2. Mutations to a glycine loop in the catalytic site of human Lon changes its protease, peptidase and ATPase activities

    Czech Academy of Sciences Publication Activity Database

    Ambro, L.; Pevala, V.; Ondrovičová, G.; Bellová, J.; Kunová, N.; Kutejová, Eva; Bauer, J.

    2014-01-01

    Roč. 281, č. 7 (2014), s. 1784-1797 ISSN 1742-464X Institutional support: RVO:61388971 Keywords : ATP-dependent protease * glycine loop * human Lon protease Subject RIV: CE - Biochemistry Impact factor: 4.001, year: 2014

  3. Expression, purification, crystallization and preliminary X-ray diffraction analysis of the aspartate transcarbamoylase domain of human CAD

    International Nuclear Information System (INIS)

    Ruiz-Ramos, Alba; Lallous, Nada; Grande-García, Araceli; Ramón-Maiques, Santiago

    2013-01-01

    The recombinant aspartate transcarbamoylase domain of human CAD was expressed in E. coli, purified and crystallized in the presence and absence of the inhibitor PALA. X-ray diffraction data sets were collected for both crystal forms at 2.1 Å resolution. Aspartate transcarbamoylase (ATCase) catalyzes the synthesis of N-carbamoyl-l-aspartate from carbamoyl phosphate and aspartate in the second step of the de novo biosynthesis of pyrimidines. In prokaryotes, the first three activities of the pathway, namely carbamoyl phosphate synthetase (CPSase), ATCase and dihydroorotase (DHOase), are encoded as distinct proteins that function independently or in noncovalent association. In animals, CPSase, ATCase and DHOase are part of a 243 kDa multifunctional polypeptide named CAD. Up-regulation of CAD is essential for normal and tumour cell proliferation. Although the structures of numerous prokaryotic ATCases have been determined, there is no structural information about any eukaryotic ATCase. In fact, the only detailed structural information about CAD is that it self-assembles into hexamers and trimers through interactions of the ATCase domains. Here, the expression, purification and crystallization of the ATCase domain of human CAD is reported. The recombinant protein, which was expressed in bacteria and purified with good yield, formed homotrimers in solution. Crystallization experiments both in the absence and in the presence of the inhibitor PALA yielded small crystals that diffracted X-rays to 2.1 Å resolution using synchrotron radiation. The crystals appeared to belong to the hexagonal space group P6 3 22, and Matthews coefficient calculation indicated the presence of one ATCase subunit per asymmetric unit, with a solvent content of 48%. However, analysis of the intensity statistics suggests a special case of the P2 1 lattice with pseudo-symmetry and possibly twinning

  4. Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

    Directory of Open Access Journals (Sweden)

    Kwangseon Jung

    Full Text Available Ultraviolet A (UVA irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate effects of aspartic acid on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs. Furthermore, we elucidated the UVA-antagonizing mechanisms of aspartic acid. The results of this study showed that aspartic acid attenuated the UVA-induced reduction of the proliferative potential and stemness of hAMSCs, as evidenced by increased proliferative activity in the 3-(4,5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide (MTT assay and upregulation of stemness-related genes OCT4, NANOG, and SOX2 in response to the aspartic acid treatment. UVA-induced reduction in the mRNA level of hypoxia-inducible factor (HIF-1α was also significantly recovered by aspartic acid. In addition, the antagonizing effects of aspartic acid against the UVA effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show that aspartic acid improves reduced stemness of hAMSCs induced by UVA and its effects are mediated by upregulation of HIF-1α via the inhibition of PGE2-cAMP signaling. In addition, aspartic acid may be used as an antagonizing agent to mitigate the effects of UVA.

  5. Antagonizing Effects of Aspartic Acid against Ultraviolet A-Induced Downregulation of the Stemness of Human Adipose Tissue-Derived Mesenchymal Stem Cells.

    Science.gov (United States)

    Jung, Kwangseon; Cho, Jae Youl; Soh, Young-Jin; Lee, Jienny; Shin, Seoung Woo; Jang, Sunghee; Jung, Eunsun; Kim, Min Hee; Lee, Jongsung

    2015-01-01

    Ultraviolet A (UVA) irradiation is responsible for a variety of changes in cell biology. The purpose of this study was to investigate effects of aspartic acid on UVA irradiation-induced damages in the stemness properties of human adipose tissue-derived mesenchymal stem cells (hAMSCs). Furthermore, we elucidated the UVA-antagonizing mechanisms of aspartic acid. The results of this study showed that aspartic acid attenuated the UVA-induced reduction of the proliferative potential and stemness of hAMSCs, as evidenced by increased proliferative activity in the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and upregulation of stemness-related genes OCT4, NANOG, and SOX2 in response to the aspartic acid treatment. UVA-induced reduction in the mRNA level of hypoxia-inducible factor (HIF)-1α was also significantly recovered by aspartic acid. In addition, the antagonizing effects of aspartic acid against the UVA effects were found to be mediated by reduced production of PGE2 through the inhibition of JNK and p42/44 MAPK. Taken together, these findings show that aspartic acid improves reduced stemness of hAMSCs induced by UVA and its effects are mediated by upregulation of HIF-1α via the inhibition of PGE2-cAMP signaling. In addition, aspartic acid may be used as an antagonizing agent to mitigate the effects of UVA.

  6. Fetal and perinatal outcomes in type 1 diabetes pregnancy: a randomized study comparing insulin aspart with human insulin in 322 subjects

    DEFF Research Database (Denmark)

    Hod, Moshe; Damm, Peter; Kaaja, Risto

    2008-01-01

    The objective of the study was a comparison of insulin aspart (IAsp) with human insulin (HI) in basal-bolus therapy with neutral protamine Hagedorn for fetal and perinatal outcomes of type 1 diabetes in pregnancy.......The objective of the study was a comparison of insulin aspart (IAsp) with human insulin (HI) in basal-bolus therapy with neutral protamine Hagedorn for fetal and perinatal outcomes of type 1 diabetes in pregnancy....

  7. 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

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

    Directory of Open Access Journals (Sweden)

    Timmerman J André B

    2006-03-01

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

  9. A novel serine protease with human fibrino(geno)lytic activities from Artocarpus heterophyllus latex.

    Science.gov (United States)

    Siritapetawee, Jaruwan; Thumanu, Kanjana; Sojikul, Punchapat; Thammasirirak, Sompong

    2012-07-01

    A protease was isolated and purified from Artocarpus heterophyllus (jackfruit) latex and designated as a 48-kDa antimicrobial protease (AMP48) in a previous publication. In this work, the enzyme was characterized for more biochemical and medicinal properties. Enzyme activity of AMP48 was strongly inhibited by phenylmethanesulfonyl fluoride and soybean trypsin inhibitor, indicating that the enzyme was a plant serine protease. The N-terminal amino acid sequences (A-Q-E-G-G-K-D-D-D-G-G) of AMP48 had no sequence similarity matches with any sequence databases of BLAST search and other plant serine protease. The secondary structure of this enzyme was composed of high α-helix (51%) and low β-sheet (9%). AMP48 had fibrinogenolytic activity with maximal activity between 55 and 60°C at pH 8. The enzyme efficiently hydrolyzed α followed by partially hydrolyzed β and γ subunits of human fibrinogen. In addition, the fibrinolytic activity was observed through the degradation products by SDS-PAGE and emphasized its activity by monitoring the alteration of secondary structure of fibrin clot after enzyme digestion using ATR-FTIR spectroscopy. This study presented the potential role to use AMP48 as antithrombotic for treatment thromboembolic disorders such as strokes, pulmonary emboli and deep vein thrombosis. Copyright © 2012 Elsevier B.V. All rights reserved.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-02-09

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

  11. Protease inhibitors enhance extracellular collagen fibril deposition in human mesenchymal stem cells.

    Science.gov (United States)

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2015-10-15

    Collagen is a widely used naturally occurring biomaterial for scaffolding, whereas mesenchymal stem cells (MSCs) represent a promising cell source in tissue engineering and regenerative medicine. It is generally known that cells are able to remodel their environment by simultaneous degradation of the scaffolds and deposition of newly synthesized extracellular matrix. Nevertheless, the interactions between MSCs and collagen biomaterials are poorly known, and the strategies enhancing the extracellular matrix deposition are yet to be defined. In this study, we aim to investigate the fate of collagen when it is in contact with MSCs and hypothesize that protease inhibition will enhance their extracellular deposition of collagen fibrils. Specifically, human MSCs (hMSCs) were exposed to fluorescence-labeled collagen with and without intracellular or extracellular protease inhibitors (or both) before tracing the collagen at both intracellular and extracellular spaces. Collagen were internalized by hMSCs and degraded intracellularly in lysosomes. In the presence of protease inhibitors, both intracellular collagen fibril growth and extracellular deposition of collagen fibrils were enhanced. Moreover, protease inhibitors work synergistically with ascorbic acid, a well-known matrix deposition-enhancing reagent, in further enhancing collagen fibril deposition at the extracellular space. These findings provide a better understanding of the interactions between hMSCs and collagen biomaterials and suggest a method to manipulate matrix remodeling and deposition of hMSCs, contributing to better scaffolding for tissue engineering and regenerative medicine.

  12. Insulin aspart in diabetic pregnancy

    DEFF Research Database (Denmark)

    Mathiesen, Elisabeth R

    2008-01-01

    in insulin requirements during pregnancy necessitate short-acting insulins for postprandial control of hyperglycemia. The fast-acting insulin analogue insulin aspart has been tested in a large, randomized trial of pregnant women with Type 1 diabetes and offers benefits in control of postprandial...... hyperglycemia with a tendency towards fewer episodes of severe hypoglycemia compared with human insulin. Treatment with insulin aspart was associated with a tendency toward fewer fetal losses and preterm deliveries than treatment with human insulin. Insulin aspart could not be detected in the fetal circulation...... and no increase in insulin antibodies was found. Thus, the use of insulin aspart in pregnancy is regarded safe....

  13. Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

    Directory of Open Access Journals (Sweden)

    Przemyslaw Glaza

    Full Text Available Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3, showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ and an N-terminally truncated HtrA3S (ΔN-HtrA3S were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.

  14. Structural and Functional Analysis of Human HtrA3 Protease and Its Subdomains.

    Science.gov (United States)

    Glaza, Przemyslaw; Osipiuk, Jerzy; Wenta, Tomasz; Zurawa-Janicka, Dorota; Jarzab, Miroslaw; Lesner, Adam; Banecki, Bogdan; Skorko-Glonek, Joanna; Joachimiak, Andrzej; Lipinska, Barbara

    2015-01-01

    Human HtrA3 protease, which induces mitochondria-mediated apoptosis, can be a tumor suppressor and a potential therapeutic target in the treatment of cancer. However, there is little information about its structure and biochemical properties. HtrA3 is composed of an N-terminal domain not required for proteolytic activity, a central serine protease domain and a C-terminal PDZ domain. HtrA3S, its short natural isoform, lacks the PDZ domain which is substituted by a stretch of 7 C-terminal amino acid residues, unique for this isoform. This paper presents the crystal structure of the HtrA3 protease domain together with the PDZ domain (ΔN-HtrA3), showing that the protein forms a trimer whose protease domains are similar to those of human HtrA1 and HtrA2. The ΔN-HtrA3 PDZ domains are placed in a position intermediate between that in the flat saucer-like HtrA1 SAXS structure and the compact pyramidal HtrA2 X-ray structure. The PDZ domain interacts closely with the LB loop of the protease domain in a way not found in other human HtrAs. ΔN-HtrA3 with the PDZ removed (ΔN-HtrA3-ΔPDZ) and an N-terminally truncated HtrA3S (ΔN-HtrA3S) were fully active at a wide range of temperatures and their substrate affinity was not impaired. This indicates that the PDZ domain is dispensable for HtrA3 activity. As determined by size exclusion chromatography, ΔN-HtrA3 formed stable trimers while both ΔN-HtrA3-ΔPDZ and ΔN-HtrA3S were monomeric. This suggests that the presence of the PDZ domain, unlike in HtrA1 and HtrA2, influences HtrA3 trimer formation. The unique C-terminal sequence of ΔN-HtrA3S appeared to have little effect on activity and oligomerization. Additionally, we examined the cleavage specificity of ΔN-HtrA3. Results reported in this paper provide new insights into the structure and function of ΔN-HtrA3, which seems to have a unique combination of features among human HtrA proteases.

  15. Saccharomyces boulardii Protease Inhibits the Effects of Clostridium difficile Toxins A and B in Human Colonic Mucosa

    Science.gov (United States)

    Castagliuolo, Ignazio; Riegler, Martin F.; Valenick, Leyla; LaMont, J. Thomas; Pothoulakis, Charalabos

    1999-01-01

    Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis. We have reported that S. boulardii inhibits C. difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I. Castagliuolo, J. T. LaMont, S. T. Nikulasson, and C. Pothoulakis, Infect. Immun. 64:5225–5232, 1996). The aim of this study was to further evaluate the role of S. boulardii protease in preventing C. difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C. difficile toxins. A polyclonal rabbit antiserum raised against purified S. boulardii serine protease inhibited by 73% the proteolytic activity present in S. boulardii conditioned medium in vitro. The anti-protease immunoglobulin G (IgG) prevented the action of S. boulardii on toxin A-induced intestinal secretion and mucosal permeability to [3H]mannitol in rat ileal loops, while control rabbit IgG had no effect. The anti-protease IgG also prevented the effects of S. boulardii protease on digestion of toxins A and B and on binding of [3H]toxin A and [3H]toxin B to purified human colonic BBM. Purified S. boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells. Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S. boulardii protease. We conclude that the protective effects of S. boulardii on C. difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease. PMID:9864230

  16. Inhibition of human lymphocyte proliferation and cleavage of interleukin-2 by Pseudomonas aeruginosa proteases

    DEFF Research Database (Denmark)

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

    1988-01-01

    This study was undertaken to determine the effect of Pseudomonas aeruginosa alkaline protease (AP) and elastase (ELA) on human lymphocyte function. AP at 50 micrograms/ml and ELA at 12 micrograms/ml caused a 50% inhibition of phytohemagglutinin-induced proliferation. There was no difference...... in the effect of proteases on CD4- and CD8-positive cells. To determine the effect of proteases on interleukin-2 (IL-2)-induced cell proliferation, the proteases and IL-2 were added to the IL-2-dependent CTLL-2 cell line. AP and ELA inhibited the proliferation of these cells. When IL-2 was added in excess......, the inhibition was partly reversed. ELA at 10 micrograms/ml cleaved IL-2, as judged by size chromatography of a reaction mixture containing 125I-labeled IL-2 and the proteases. The ELA-digested IL-2 exhibited a reduced binding capacity to IL-2 receptors on the lymphocytes. Furthermore, treatment...

  17. The role and molecular mechanism of D-aspartic acid in the release and synthesis of LH and testosterone in humans and rats

    Directory of Open Access Journals (Sweden)

    Ronsini Salvatore

    2009-10-01

    Full Text Available Abstract Background D-aspartic acid is an amino acid present in neuroendocrine tissues of invertebrates and vertebrates, including rats and humans. Here we investigated the effect of this amino acid on the release of LH and testosterone in the serum of humans and rats. Furthermore, we investigated the role of D-aspartate in the synthesis of LH and testosterone in the pituitary and testes of rats, and the molecular mechanisms by which this amino acid triggers its action. Methods For humans: A group of 23 men were given a daily dose of D-aspartate (DADAVIT® for 12 days, whereas another group of 20 men were given a placebo. For rats: A group of 10 rats drank a solution of either 20 mM D-aspartate or a placebo for 12 days. Then LH and testosterone accumulation was determined in the serum and D-aspartate accumulation in tissues. The effects of D-aspartate on the synthesis of LH and testosterone were gauged on isolated rat pituitary and Leydig cells. Tissues were incubated with D-aspartate, and then the concentration (synthesis of LH and cGMP in the pituitary and of testosterone and cAMP in the Leydig cells was determined. Results In humans and rats, sodium D-aspartate induces an enhancement of LH and testosterone release. In the rat pituitary, sodium D-aspartate increases the release and synthesis of LH through the involvement of cGMP as a second messenger, whereas in rat testis Leydig cells, it increases the synthesis and release of testosterone and cAMP is implicated as second messenger. In the pituitary and in testes D-Asp is synthesized by a D-aspartate racemase which convert L-Asp into D-Asp. The pituitary and testes possesses a high capacity to trapping circulating D-Asp from hexogen or endogen sources. Conclusion D-aspartic acid is a physiological amino acid occurring principally in the pituitary gland and testes and has a role in the regulation of the release and synthesis of LH and testosterone in humans and rats.

  18. Human mast cell tryptase: Multiple cDNAs and genes reveal a multigene serine protease family

    International Nuclear Information System (INIS)

    Vanderslice, P.; Ballinger, S.M.; Tam, E.K.; Goldstein, S.M.; Craik, C.S.; Caughey, G.H.

    1990-01-01

    Three different cDNAs and a gene encoding human skin mast cell tryptase have been cloned and sequenced in their entirety. The deduced amino acid sequences reveal a 30-amino acid prepropeptide followed by a 245-amino acid catalytic domain. The C-terminal undecapeptide of the human preprosequence is identical in dog tryptase and appears to be part of a prosequence unique among serine proteases. The differences among the three human tryptase catalytic domains include the loss of a consensus N-glycosylation site in one cDNA, which may explain some of the heterogeneity in size and susceptibility to deglycosylation seen in tryptase preparations. All three tryptase cDNAs are distinct from a recently reported cDNA obtained from a human lung mast cell library. A skin tryptase cDNA was used to isolate a human tryptase gene, the exons of which match one of the skin-derived cDNAs. The organization of the ∼1.8-kilobase-pair tryptase gene is unique and is not closely related to that of any other mast cell or leukocyte serine protease. The 5' regulatory regions of the gene share features with those of other serine proteases, including mast cell chymase, but are unusual in being separated from the protein-coding sequence by an intron. High-stringency hybridization of a human genomic DNA blot with a fragment of the tryptase gene confirms the presence of multiple tryptase genes. These findings provide genetic evidence that human mast cell tryptases are the products of a multigene family

  19. Positive selection pressure introduces secondary mutations at Gag cleavage sites in human immunodeficiency virus type 1 harboring major protease resistance mutations

    DEFF Research Database (Denmark)

    Banke, S.; Lillemark, M.R.; Gerstoft, J.

    2009-01-01

    Human immunodeficiency virus type 1 (HIV-1) protease inhibitors (PIs) specifically target the HIV-1 protease enzyme. Mutations in the enzyme can result in PI resistance (termed PI mutations); however, mutations in the HIV-1 gag region, the substrate for the protease enzyme, might also lead to PI ...

  20. Primary structure of human pancreatic protease E determined by sequence analysis of the cloned mRNA

    International Nuclear Information System (INIS)

    Shen, W.; Fletcher, T.S.; Largman, C.

    1987-01-01

    Although protease E was isolated from human pancreas over 10 years ago, its amino acid sequence and relationship to the elastases have not been established. The authors report the isolation of a cDNA clone for human pancreatic protease E and determination of the nucleic acid sequence coding for the protein. The deduced amino acid sequence contains all of the features common to serine proteases. The substrate binding region is highly homologous to those of porcine and rat elastases 1, explaining the similar specificity for alanine reported for protease E and these elastases. However, the amino acid sequence outside the substrate binding region is less than 50% conserved, and there is a striking difference in the overall net charge for protease E (6-) and elastases 1 (8+). These findings confirm that protease E is a new member of the serine protease family. They have attempted to identify amino acid residues important for the interaction between elastases and elastin by examining the amino acid sequence differences between elastases and protease E. In addition to the large number of surface charge changes which are outside the substrate binding region, there are several changes which might be crucial for elastolysis: Leu-73/Arg-73; Arg-217A/Ala-217A; Arg-65A/Gln-65A; and the presence of two new cysteine residues (Cys-98 and Cys-99B) which computer modeling studies predict could form a new disulfide bond, not previously observed for serine proteases. They also present evidence which suggests that human pancreas does not synthesize a basic, alanine-specific elastase similar to porcine elastase 1

  1. Targeting cysteine residues of human immunodeficiency virus type 1 protease by reactive free radical species.

    Science.gov (United States)

    Basu, A; Sehajpal, P K; Ogiste, J S; Lander, H M

    1999-01-01

    Nitric oxide (NO) is a naturally occurring free radical with many functions. The oxidized form of NO, the nitrosonium ion, reacts with the thiol group of cysteine residues resulting in their modification to S-nitrosothiols. The human immunodeficiency virus type 1 (HIV-1) protease (HIV-PR) has two cysteine residues that are conserved amongst different viral isolates found in patients with acquired immunodeficiency syndrome (AIDS). In an active dimer, these residues are located near the surface of the protease. We have found that treatment of HIV-PR with different NO congeners results in loss of its proteolytic activity and simultaneous formation of S-nitrosothiols. Sodium nitroprusside inhibited HIV-PR up to 70% and S-nitroso-N-acetylpenicillamine completely inhibited the protease within 5 min of treatment. The pattern of inhibition by NO donors is comparable to its inhibition by N-acetyl pepstatin. Using electrospray ionization-mass spectrometry, we identified the modification of HIV-PR by NO as that of S-nitrosation. Our findings point towards a possible role of NO in mediating resistance to HIV-1 infection.

  2. Production of extracellular aspartic protease in submerged ...

    African Journals Online (AJOL)

    Fungal milk-clotting enzymes have gained value as bovine Chymosin substitutes in the cheese industry. In this work, the effects of culture conditions on the production of extracellular milk clotting enzymes from Mucor mucedo DSM 809 in submerged fermentation were studied. The maximum activity was observed after 48 h ...

  3. 21 CFR 582.5017 - Aspartic acid.

    Science.gov (United States)

    2010-04-01

    ... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Aspartic acid. 582.5017 Section 582.5017 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) ANIMAL DRUGS... 1 § 582.5017 Aspartic acid. (a) Product. Aspartic acid (L- and DL-forms). (b) Conditions of use...

  4. Human Kunitz-type protease inhibitor engineered for enhanced matrix retention extends longevity of fibrin biomaterials.

    Science.gov (United States)

    Briquez, Priscilla S; Lorentz, Kristen M; Larsson, Hans M; Frey, Peter; Hubbell, Jeffrey A

    2017-08-01

    Aprotinin is a broad-spectrum serine protease inhibitor used in the clinic as an anti-fibrinolytic agent in fibrin-based tissue sealants. However, upon re-exposure, some patients suffer from hypersensitivity immune reactions likely related to the bovine origin of aprotinin. Here, we aimed to develop a human-derived substitute to aprotinin. Based on sequence homology analyses, we identified the Kunitz-type protease inhibitor (KPI) domain of human amyloid-β A4 precursor protein as being a potential candidate. While KPI has a lower intrinsic anti-fibrinolytic activity than aprotinin, we reasoned that its efficacy is additionally limited by its fast release from fibrin material, just as aprotinin's is. Thus, we engineered KPI variants for controlled retention in fibrin biomaterials, using either covalent binding through incorporation of a substrate for the coagulation transglutaminase Factor XIIIa or through engineering of extracellular matrix protein super-affinity domains for sequestration into fibrin. We showed that both engineered KPI variants significantly slowed plasmin-mediated fibrinolysis in vitro, outperforming aprotinin. In vivo, our best engineered KPI variant (incorporating the transglutaminase substrate) extended fibrin matrix longevity by 50%, at a dose at which aprotinin did not show efficacy, thus qualifying it as a competitive substitute of aprotinin in fibrin sealants. Copyright © 2017 The Author(s). Published by Elsevier Ltd.. All rights reserved.

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

  6. Structure and Functional Characterization of Human Aspartate Transcarbamoylase, the Target of the Anti-tumoral Drug PALA.

    Science.gov (United States)

    Ruiz-Ramos, Alba; Velázquez-Campoy, Adrián; Grande-García, Araceli; Moreno-Morcillo, María; Ramón-Maiques, Santiago

    2016-07-06

    CAD, the multienzymatic protein that initiates and controls de novo synthesis of pyrimidines in animals, associates through its aspartate transcarbamoylase (ATCase) domain into particles of 1.5 MDa. Despite numerous structures of prokaryotic ATCases, we lack structural information on the ATCase domain of CAD. Here, we report the structure and functional characterization of human ATCase, confirming the overall similarity with bacterial homologs. Unexpectedly, human ATCase exhibits cooperativity effects that reduce the affinity for the anti-tumoral drug PALA. Combining structural, mutagenic, and biochemical analysis, we identified key elements for the necessary regulation and transmission of conformational changes leading to cooperativity between subunits. Mutation of one of these elements, R2024, was recently found to cause the first non-lethal CAD deficit. We reproduced this mutation in human ATCase and measured its effect, demonstrating that this arginine is part of a molecular switch that regulates the equilibrium between low- and high-affinity states for the ligands. Copyright © 2016 Elsevier Ltd. All rights reserved.

  7. Host-Parasite Interaction: Parasite-Derived and -Induced Proteases That Degrade Human Extracellular Matrix

    Directory of Open Access Journals (Sweden)

    Carolina Piña-Vázquez

    2012-01-01

    Full Text Available Parasitic protozoa are among the most important pathogens worldwide. Diseases such as malaria, leishmaniasis, amoebiasis, giardiasis, trichomoniasis, and trypanosomiasis affect millions of people. Humans are constantly threatened by infections caused by these pathogens. Parasites engage a plethora of surface and secreted molecules to attach to and enter mammalian cells. The secretion of lytic enzymes by parasites into host organs mediates critical interactions because of the invasion and destruction of interstitial tissues, enabling parasite migration to other sites within the hosts. Extracellular matrix is a complex, cross-linked structure that holds cells together in an organized assembly and that forms the basement membrane lining (basal lamina. The extracellular matrix represents a major barrier to parasites. Therefore, the evolution of mechanisms for connective-tissue degradation may be of great importance for parasite survival. Recent advances have been achieved in our understanding of the biochemistry and molecular biology of proteases from parasitic protozoa. The focus of this paper is to discuss the role of protozoan parasitic proteases in the degradation of host ECM proteins and the participation of these molecules as virulence factors. We divide the paper into two sections, extracellular and intracellular protozoa.

  8. Neuropeptidomics Mass Spectrometry Reveals Signaling Networks Generated by Distinct Protease Pathways in Human Systems

    Science.gov (United States)

    Hook, Vivian; Bandeira, Nuno

    2015-12-01

    Neuropeptides regulate intercellular signaling as neurotransmitters of the central and peripheral nervous systems, and as peptide hormones in the endocrine system. Diverse neuropeptides of distinct primary sequences of various lengths, often with post-translational modifications, coordinate and integrate regulation of physiological functions. Mass spectrometry-based analysis of the diverse neuropeptide structures in neuropeptidomics research is necessary to define the full complement of neuropeptide signaling molecules. Human neuropeptidomics has notable importance in defining normal and dysfunctional neuropeptide signaling in human health and disease. Neuropeptidomics has great potential for expansion in translational research opportunities for defining neuropeptide mechanisms of human diseases, providing novel neuropeptide drug targets for drug discovery, and monitoring neuropeptides as biomarkers of drug responses. In consideration of the high impact of human neuropeptidomics for health, an observed gap in this discipline is the few published articles in human neuropeptidomics compared with, for example, human proteomics and related mass spectrometry disciplines. Focus on human neuropeptidomics will advance new knowledge of the complex neuropeptide signaling networks participating in the fine control of neuroendocrine systems. This commentary review article discusses several human neuropeptidomics accomplishments that illustrate the rapidly expanding diversity of neuropeptides generated by protease processing of pro-neuropeptide precursors occurring within the secretory vesicle proteome. Of particular interest is the finding that human-specific cathepsin V participates in producing enkephalin and likely other neuropeptides, indicating unique proteolytic mechanisms for generating human neuropeptides. The field of human neuropeptidomics has great promise to solve new mechanisms in disease conditions, leading to new drug targets and therapeutic agents for human

  9. Glycolipid-Dependent, Protease Sensitive Internalization of Pseudomonas aeruginosa Into Cultured Human Respiratory Epithelial Cells

    Science.gov (United States)

    Emam, Aufaugh; Carter, William G; Lingwood, Clifford

    2010-01-01

    Internalization of PAK strain Pseudomonas aeruginosa into human respiratory epithelial cell lines and HeLa cervical cancer cells in vitro was readily demonstrable via a gentamycin protection assay. Depletion of target cell glycosphingolipids (GSLs) using a glucosyl ceramide synthase inhibitor, P4, completely prevented P. aeruginosa internalization. In contrast, P4 treatment had no effect on the internalization of Salmonella typhimurium into HeLa cells. Internalized P. aeruginosa were within membrane vacuoles, often containing microvesicles, between the bacterium and the limiting membrane. P. aeruginosa internalization was markedly enhanced by target cell pretreatment with the exogenous GSL, deacetyl gangliotetraosyl ceramide (Gg4). Gg4 binds the lipid raft marker, GM1 ganglioside. Target cell pretreatment with TLCK, but not other (serine) protease inhibitors, prevented both P. aeruginosa host cell binding and internalization. NFkB inhibition also prevented internalization. A GSL-containing lipid-raft model of P. aeruginosa host cell binding/internalization is proposed PMID:21270937

  10. Crystal Structure of Human Taspase1, a Crucial Protease Regulating the Function of MLL

    Energy Technology Data Exchange (ETDEWEB)

    Khan,J.; Dunn, B.; Tong, L.

    2005-01-01

    Taspase1 catalyzes the proteolytic processing of the mixed lineage leukemia (MLL) nuclear protein, which is required for maintaining Hox gene expression patterns. Chromosomal translocations of the MLL gene are associated with leukemia in infants. Taspase1, a threonine aspartase, is a member of the type 2 asparaginase family, but is the only protease in this family. We report here the crystal structures of human activated Taspase1 and its proenzyme, as well as the characterization of the effects of mutations in the active site region using a newly developed fluorogenic assay. The structure of Taspase1 has significant differences from other asparaginases, especially near the active site. Mutation of the catalytic nucleophile, Thr234, abolishes autocatalytic processing in cis but does not completely block proteolysis in trans. The structure unexpectedly showed the binding of a chloride ion in the active site, and our kinetic studies confirm that chlorides ions are inhibitors of this enzyme at physiologically relevant concentrations.

  11. Position of the third Na+ site in the aspartate transporter GltPh and the human glutamate transporter, EAAT1.

    Directory of Open Access Journals (Sweden)

    Turgut Bastug

    Full Text Available Glutamate transport via the human excitatory amino acid transporters is coupled to the co-transport of three Na(+ ions, one H(+ and the counter-transport of one K(+ ion. Transport by an archaeal homologue of the human glutamate transporters, Glt(Ph, whose three dimensional structure is known is also coupled to three Na(+ ions but only two Na(+ ion binding sites have been observed in the crystal structure of Glt(Ph. In order to fully utilize the Glt(Ph structure in functional studies of the human glutamate transporters, it is essential to understand the transport mechanism of Glt(Ph and accurately determine the number and location of Na(+ ions coupled to transport. Several sites have been proposed for the binding of a third Na(+ ion from electrostatic calculations and molecular dynamics simulations. In this study, we have performed detailed free energy simulations for Glt(Ph and reveal a new site for the third Na(+ ion involving the side chains of Threonine 92, Serine 93, Asparagine 310, Aspartate 312, and the backbone of Tyrosine 89. We have also studied the transport properties of alanine mutants of the coordinating residues Threonine 92 and Serine 93 in Glt(Ph, and the corresponding residues in a human glutamate transporter, EAAT1. The mutant transporters have reduced affinity for Na(+ compared to their wild type counterparts. These results confirm that Threonine 92 and Serine 93 are involved in the coordination of the third Na(+ ion in Glt(Ph and EAAT1.

  12. Longevity of elastin in human intervertebral disc as probed by the racemization of aspartic acid

    DEFF Research Database (Denmark)

    Sivan, Sarit-Sara; Van El, Benno; Merkher, Yulia

    2012-01-01

    BACKGROUND: Aging and degeneration of human intervertebral disc (IVD) are associated with biochemical changes, including racemization and glycation. These changes can only be counteracted by protein turnover. Little is known about the longevity of IVD elastin in health or disease. Yet, such knowl...

  13. Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism.

    Science.gov (United States)

    Lee, Young Ah; Nam, Young Hee; Min, Arim; Kim, Kyeong Ah; Nozaki, Tomoyoshi; Saito-Nakano, Yumiko; Mirelman, David; Shin, Myeong Heon

    2014-01-01

    Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs) contain large amounts of cysteine proteases (CPs), one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells) were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP)-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2) did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis. © Y.A. Lee et al., published by EDP Sciences, 2014.

  14. Entamoeba histolytica-secreted cysteine proteases induce IL-8 production in human mast cells via a PAR2-independent mechanism

    Directory of Open Access Journals (Sweden)

    Lee Young Ah

    2014-01-01

    Full Text Available Entamoeba histolytica is an extracellular tissue parasite causing colitis and occasional liver abscess in humans. E. histolytica-derived secretory products (SPs contain large amounts of cysteine proteases (CPs, one of the important amoebic virulence factors. Although tissue-residing mast cells play an important role in the mucosal inflammatory response to this pathogen, it is not known whether the SPs induce mast cell activation. In this study, when human mast cells (HMC-1 cells were stimulated with SPs collected from pathogenic wild-type amoebae, interleukin IL-8 mRNA expression and production were significantly increased compared with cells incubated with medium alone. Inhibition of CP activity in the SPs with heat or the CP inhibitor E64 resulted in significant reduction of IL-8 production. Moreover, SPs obtained from inhibitors of cysteine protease (ICP-overexpressing amoebae with low CP activity showed weaker stimulatory effects on IL-8 production than the wild-type control. Preincubation of HMC-1 cells with antibodies to human protease-activated receptor 2 (PAR2 did not affect the SP-induced IL-8 production. These results suggest that cysteine proteases in E. histolytica-derived secretory products stimulate mast cells to produce IL-8 via a PAR2-independent mechanism, which contributes to IL-8-mediated tissue inflammatory responses during the early phase of human amoebiasis.

  15. Safety, pharmacokinetics, and antiviral activity of A77003, a C2 symmetry-based human immunodeficiency virus protease inhibitor

    NARCIS (Netherlands)

    Reedijk, M.; Boucher, C. A.; van Bommel, T.; Ho, D. D.; Tzeng, T. B.; Sereni, D.; Veyssier, P.; Jurriaans, S.; Granneman, R.; Hsu, A.

    1995-01-01

    A77003, an inhibitor of the human immunodeficiency virus type 1 (HIV-1) protease, was administered to asymptomatic HIV-1-infected patients in a phase I trial. The drug was given by continuous intravenous infusion at dosages of 0.035, 0.07, 0.14, and 0.28 mg/kg of body weight per h. The drug was

  16. Fusion to Human Serum Albumin Extends the Circulatory Half-Life and Duration of Antithrombotic Action of the Kunitz Protease Inhibitor Domain of Protease Nexin 2.

    Science.gov (United States)

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

    2018-01-01

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

  17. 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.

  18. Protease-activated receptor 2 agonist increases cell proliferation and invasion of human pancreatic cancer cells

    Science.gov (United States)

    XIE, LIQUN; DUAN, ZEXING; LIU, CAIJU; ZHENG, YANMIN; ZHOU, JING

    2015-01-01

    The aim of this study was to determine the expression of protease-activated receptor 2 (PAR-2) in the human pancreatic cancer cell line SW1990, and to evaluate its effect on cell proliferation and invasion. The expression of PAR-2 protein and mRNA in SW1990 cells was determined by immunocytochemistry and reverse transcription polymerase chain reaction (PCR), respectively. MTT and cell invasion and migration assays, as well as semi-quantitative PCR and zymography analysis, were additionally performed. PAR-2 mRNA was significantly upregulated in the cells treated with trypsin or the PAR-2 activating peptide Ser-Leu-Ile-Gly-Lys-Val (SLIGKV) (P0.05). Trypsin and SLIGKV significantly promoted SW1990 cell proliferation in a dose- and time-dependent manner (P<0.05). Compared with the control group, trypsin and SLIGKV significantly increased the mRNA expression (P<0.01) and gelatinolytic activity (P<0.01) of matrix metalloproteinase (MMP)-2. In conclusion, PAR-2 is expressed in SW1990 cells. PAR-2 activation may promote the invasion and migration of human pancreatic cancer cells by increasing MMP-2 expression. PMID:25452809

  19. Herpes simplex virus downregulation of secretory leukocyte protease inhibitor enhances human papillomavirus type 16 infection.

    Science.gov (United States)

    Skeate, Joseph G; Porras, Tania B; Woodham, Andrew W; Jang, Julie K; Taylor, Julia R; Brand, Heike E; Kelly, Thomas J; Jung, Jae U; Da Silva, Diane M; Yuan, Weiming; Kast, W Martin

    2016-02-01

    Herpes simplex virus (HSV) was originally implicated in the aetiology of cervical cancer, and although high-risk human papillomavirus (HPV) is now the accepted causative agent, the epidemiological link between HSV and HPV-associated cancers persists. The annexin A2 heterotetramer (A2t) has been shown to mediate infectious HPV type 16 (HPV16) uptake by human keratinocytes, and secretory leukocyte protease inhibitor (SLPI), an endogenous A2t ligand, inhibits HPV16 uptake and infection. Interestingly, HSV infection induces a sustained downregulation of SLPI in epithelial cells, which we hypothesized promotes HPV16 infection through A2t. Here, we show that in vitro infection of human keratinocytes with HSV-1 or HSV-2, but not with an HSV-1 ICP4 deletion mutant that does not downregulate SLPI, leads to a >70% reduction of SLPI mRNA and a >60% decrease in secreted SLPI protein. Consequently, we observed a significant increase in the uptake of HPV16 virus-like particles and gene transduction by HPV16 pseudovirions (two- and 2.5-fold, respectively) in HSV-1- and HSV-2-infected human keratinocyte cell cultures compared with uninfected cells, whereas exogenously added SLPI reversed this effect. Using a SiMPull (single-molecule pulldown) assay, we demonstrated that endogenously secreted SLPI interacts with A2t on epithelial cells in an autocrine/paracrine manner. These results suggested that ongoing HSV infection and resultant downregulation of local levels of SLPI may impart a greater susceptibility for keratinocytes to HPV16 infection through the host cell receptor A2t, providing a mechanism that may, in part, provide an explanation for the aetiological link between HSV and HPV-associated cancers.

  20. MicroRNA regulation of human protease genes essential for influenza virus replication.

    Directory of Open Access Journals (Sweden)

    Victoria A Meliopoulos

    Full Text Available Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB, cAMP/calcium signaling (CRE/CREB, and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

  1. MicroRNA regulation of human protease genes essential for influenza virus replication.

    Science.gov (United States)

    Meliopoulos, Victoria A; Andersen, Lauren E; Brooks, Paula; Yan, Xiuzhen; Bakre, Abhijeet; Coleman, J Keegan; Tompkins, S Mark; Tripp, Ralph A

    2012-01-01

    Influenza A virus causes seasonal epidemics and periodic pandemics threatening the health of millions of people each year. Vaccination is an effective strategy for reducing morbidity and mortality, and in the absence of drug resistance, the efficacy of chemoprophylaxis is comparable to that of vaccines. However, the rapid emergence of drug resistance has emphasized the need for new drug targets. Knowledge of the host cell components required for influenza replication has been an area targeted for disease intervention. In this study, the human protease genes required for influenza virus replication were determined and validated using RNA interference approaches. The genes validated as critical for influenza virus replication were ADAMTS7, CPE, DPP3, MST1, and PRSS12, and pathway analysis showed these genes were in global host cell pathways governing inflammation (NF-κB), cAMP/calcium signaling (CRE/CREB), and apoptosis. Analyses of host microRNAs predicted to govern expression of these genes showed that eight miRNAs regulated gene expression during virus replication. These findings identify unique host genes and microRNAs important for influenza replication providing potential new targets for disease intervention strategies.

  2. The human immunodeficiency virus protease inhibitor ritonavir is potentially active against urological malignancies

    Directory of Open Access Journals (Sweden)

    Sato A

    2015-04-01

    Full Text Available Akinori Sato Department of Urology, National Defense Medical College, Tokorozawa, Japan Abstract: The human immunodeficiency virus protease inhibitor ritonavir has recently been shown to have antineoplastic activity, and its use in urological malignancies is under investigation with an eye toward drug repositioning. Ritonavir is thought to exert its antineoplastic activity by inhibiting multiple signaling pathways, including the Akt and nuclear factor-kappaB pathways. It can increase the amount of unfolded proteins in the cell by inhibiting both the proteasome and heat shock protein 90. Combinations of ritonavir with agents that increase the amount of unfolded proteins, such as proteasome inhibitors, histone deacetylase inhibitors, or heat shock protein 90 inhibitors, therefore, induce endoplasmic reticulum stress cooperatively and thereby kill cancer cells effectively. Ritonavir is also a potent cytochrome P450 3A4 and P-glycoprotein inhibitor, increasing the intracellular concentration of combined drugs by inhibiting their degradation and efflux from cancer cells and thereby enhancing their antineoplastic activity. Furthermore, riotnavir’s antineoplastic activity includes modulation of immune system activity. Therapies using ritonavir are thus an attractive new approach to cancer treatment and, due to their novel mechanisms of action, are expected to be effective against malignancies that are refractory to current treatment strategies. Further investigations using ritonavir are expected to find new uses for clinically available drugs in the treatment of urological malignancies as well as many other types of cancer. Keywords: drug repositioning, novel treatment

  3. Leishmania major surface protease Gp63 interferes with the function of human monocytes and neutrophils in vitro

    DEFF Research Database (Denmark)

    Sørensen, A L; Hey, A S; Kharazmi, A

    1994-01-01

    In the present study the effect of Leishmania major surface protease Gp63 on the chemotaxis and oxidative burst response of human peripheral blood monocytes and neutrophils was investigated. It was shown that prior incubation of cells with Gp63 inhibited chemotaxis of neutrophils but not monocytes...... towards the chemotactic peptide f-met-leu-phe. On the other hand, chemotaxis of both neutrophils and monocytes towards zymosan-activated serum containing C5a was inhibited by Gp63. Monocyte and neutrophil chemiluminescence response to opsonized zymosan was reduced by preincubation of the cells with Gp63...... in a concentration-dependent manner. Notably, monocytes were inhibited to a much greater degree than neutrophils by a given concentration of Gp63, and they were also inhibited at much lower concentrations of the protease. The inhibitory effect of Gp63 on chemotaxis and chemiluminescence was completely abolished...

  4. Marked longevity of human lung parenchymal elastic fibers deduced from prevalence of D-aspartate and nuclear weapons-related radiocarbon

    International Nuclear Information System (INIS)

    Shapiro, S.D.; Endicott, S.K.; Province, M.A.; Pierce, J.A.; Campbell, E.J.

    1991-01-01

    Normal structure and function of the lung parenchyma depend upon elastic fibers. Amorphous elastin is biochemically stable in vitro, and may provide a metabolically stable structural framework for the lung parenchyma. To test the metabolic stability of elastin in the normal human lung parenchyma, we have (a) estimated the time elapsed since the synthesis of the protein through measurement of aspartic acid racemization and (b) modeled the elastin turnover through measurement of the prevalence of nuclear weapons-related 14 C. Elastin purified by a new technique from normal lung parenchyma was hydrolyzed; then the prevalences of D-aspartate and 14 C were measured by gas chromatography and accelerator-mass spectrometry, respectively. D-aspartate increased linearly with age; Kasp (1.76 x 10 - 3 yr - 1 ) was similar to that previously found for extraordinarily stable human tissues, indicating that the age of lung parenchymal elastin corresponded with the age of the subject. Radiocarbon prevalence data also were consistent with extraordinary metabolic stability of elastin; the calculated mean carbon residence time in elastin was 74 yr (95% confidence limits, 40-174 yr). These results indicate that airspace enlargement characteristic of 'aging lung' is not associated with appreciable new synthesis of lung parenchymal elastin. The present study provides the first tissue-specific evaluation of turnover of an extracellular matrix component in humans and underscores the potential importance of elastin for maintenance of normal lung structure. Most importantly, the present work provides a foundation for strategies to directly evaluate extracellular matrix injury and repair in diseases of lung (especially pulmonary emphysema), vascular tissue, and skin

  5. Insulin aspart pharmacokinetics

    DEFF Research Database (Denmark)

    Rasmussen, Christian Hove; Roge, Rikke Meldgaard; Ma, Zhulin

    2014-01-01

    Background: Insulin aspart (IAsp) is used by many diabetics as a meal-time insulin to control postprandial glucose levels. As is the case with many other insulin types, the pharmacokinetics (PK), and consequently the pharmacodynamics (PD), is associated with clinical variability, both between...... to investigate and quantify the properties of the subcutaneous depot. Data from Brange et al. (1990) are used to determine the effects of insulin chemistry in subcutis on the absorption rate. Intravenous (i.v.) bolus and infusion PK data for human insulin are used to understand and quantify the systemic...... distribution and elimination (Porksen et al., 1997; Sjostrand et al., 2002). PK and PD profiles for type 1 diabetics from Chen et al. (2005) are analyzed to demonstrate the effects of IAsp antibodies in terms of bound and unbound insulin. PK profiles from Thorisdottir et al. (2009) and Ma et al. (2012b...

  6. Effect of Legionella pneumophila cytotoxic protease on human neutrophil and monocyte function

    DEFF Research Database (Denmark)

    Rechnitzer, C; Kharazmi, A

    1992-01-01

    The extracellular metalloprotease of Legionella pneumophila, also called tissue-destructive protease or major secretory protein, has been proposed as one of the virulence factors of this organism. Considering the decisive role played by the phagocytic cells in host defense against Legionella...

  7. Characterization of a serine protease-mediated cell death program activated in human leukemia cells

    International Nuclear Information System (INIS)

    O'Connell, A.R.; Holohan, C.; Torriglia, A.; Lee, B.F.; Stenson-Cox, C.

    2006-01-01

    Tightly controlled proteolysis is a defining feature of apoptosis and caspases are critical in this regard. Significant roles for non-caspase proteases in cell death have been highlighted. Staurosporine causes a rapid induction of apoptosis in virtually all mammalian cell types. Numerous studies demonstrate that staurosporine can activate cell death under caspase-inhibiting circumstances. The aim of this study was to investigate the proteolytic mechanisms responsible for cell death under these conditions. To that end, we show that inhibitors of serine proteases can delay cell death in one such system. Furthermore, through profiling of proteolytic activation, we demonstrate, for the first time, that staurosporine activates a chymotrypsin-like serine protease-dependent cell death in HL-60 cells independently, but in parallel with the caspase controlled systems. Features of the serine protease-mediated system include cell shrinkage and apoptotic morphology, regulation of caspase-3, altered nuclear morphology, generation of an endonuclease and DNA degradation. We also demonstrate a staurosporine-induced activation of a putative 16 kDa chymotrypsin-like protein during apoptosis

  8. Structural and functional characterization of cleavage and inactivation of human serine protease inhibitors by the bacterial SPATE protease EspPα from enterohemorrhagic E. coli.

    Directory of Open Access Journals (Sweden)

    André Weiss

    Full Text Available EspPα and EspI are serine protease autotransporters found in enterohemorrhagic Escherichia coli. They both belong to the SPATE autotransporter family and are believed to contribute to pathogenicity via proteolytic cleavage and inactivation of different key host proteins during infection. Here, we describe the specific cleavage and functional inactivation of serine protease inhibitors (serpins by EspPα and compare this activity with the related SPATE EspI. Serpins are structurally related proteins that regulate vital protease cascades, such as blood coagulation and inflammatory host response. For the rapid determination of serpin cleavage sites, we applied direct MALDI-TOF-MS or ESI-FTMS analysis of coincubations of serpins and SPATE proteases and confirmed observed cleavage positions using in-gel-digest of SDS-PAGE-separated degradation products. Activities of both serpin and SPATE protease were assessed in a newly developed photometrical assay using chromogenic peptide substrates. EspPα cleaved the serpins α1-protease inhibitor (α1-PI, α1-antichymotrypsin, angiotensinogen, and α2-antiplasmin. Serpin cleavage led to loss of inhibitory function as demonstrated for α1-PI while EspPα activity was not affected. Notably, EspPα showed pronounced specificity and cleaved procoagulatory serpins such as α2-antiplasmin while the anticoagulatory antithrombin III was not affected. Together with recently published research, this underlines the interference of EspPα with hemostasis or inflammatory responses during infection, while the observed interaction of EspI with serpins is likely to be not physiologically relevant. EspPα-mediated serpin cleavage occurred always in flexible loops, indicating that this structural motif might be required for substrate recognition.

  9. Immunological changes in human immunodeficiency virus (HIV)-infected individuals during HIV-specific protease inhibitor treatment

    DEFF Research Database (Denmark)

    Ullum, H; Katzenstein, T; Aladdin, H

    1999-01-01

    The present study examines the influence of effective anti-retroviral treatment on immune function, evaluated by a broad array of immunological tests. We followed 12 individuals infected with human immunodeficiency virus (HIV) for 6 months after initiation of combination anti-retroviral treatment...... including a protease inhibitor. Unstimulated and pokeweed mitogen (PWM)-, interleukin (IL)-2- and phytohaemagglutinin (PHA)-stimulated lymphocyte proliferative responses increased during follow-up reaching average levels from 1.3-fold (PHA) to 3.7-fold (PWM) above baseline values. The total CD4+ lymphocyte...

  10. Cell-based fluorescence assay for human immunodeficiency virus type 1 protease activity

    Czech Academy of Sciences Publication Activity Database

    Lindsten, K.; Kondrová, Taťána; Konvalinka, Jan; Masucci, M. G.; Dantuma, N. P.

    2001-01-01

    Roč. 45, č. 9 (2001), s. 2616-2622 ISSN 0066-4804 R&D Projects: GA ČR GA303/98/1559 Grant - others:HHMI(US) 75195-540801; ECTM(XE) ERBFMRXCT960026 Institutional research plan: CEZ:AV0Z4055905 Keywords : HIV-1 * protease activity Subject RIV: CE - Biochemistry Impact factor: 4.562, year: 2001

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

    Directory of Open Access Journals (Sweden)

    Jan Dvorák

    2009-06-01

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

  12. Three-Dimensional Reconstruction of the S885A Mutant of Human Mitochondrial Lon Protease

    Czech Academy of Sciences Publication Activity Database

    Kereiche, S.; Kováčik, L.; Pevala, V.; Ambro, L.; Bellová, J.; Kutejová, Eva; Raška, I.

    2014-01-01

    Roč. 60, č. 2014 (2014), s. 62-65 ISSN 0015-5632 R&D Projects: GA MŠk(CZ) EE2.3.30.0030; GA MŠk(CZ) ED1.1.00/02.0109 Institutional support: RVO:61388971 Keywords : transmission electron microscopy * 3D reconstruction * AAA plus protease Subject RIV: CE - Biochemistry Impact factor: 1.000, year: 2014

  13. The C-terminal sequence of several human serine proteases encodes host defense functions.

    Science.gov (United States)

    Kasetty, Gopinath; Papareddy, Praveen; Kalle, Martina; Rydengård, Victoria; Walse, Björn; Svensson, Bo; Mörgelin, Matthias; Malmsten, Martin; Schmidtchen, Artur

    2011-01-01

    Serine proteases of the S1 family have maintained a common structure over an evolutionary span of more than one billion years, and evolved a variety of substrate specificities and diverse biological roles, involving digestion and degradation, blood clotting, fibrinolysis and epithelial homeostasis. We here show that a wide range of C-terminal peptide sequences of serine proteases, particularly from the coagulation and kallikrein systems, share characteristics common with classical antimicrobial peptides of innate immunity. Under physiological conditions, these peptides exert antimicrobial effects as well as immunomodulatory functions by inhibiting macrophage responses to bacterial lipopolysaccharide. In mice, selected peptides are protective against lipopolysaccharide-induced shock. Moreover, these S1-derived host defense peptides exhibit helical structures upon binding to lipopolysaccharide and also permeabilize liposomes. The results uncover new and fundamental aspects on host defense functions of serine proteases present particularly in blood and epithelia, and provide tools for the identification of host defense molecules of therapeutic interest. Copyright © 2011 S. Karger AG, Basel.

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

    DEFF Research Database (Denmark)

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

    2006-01-01

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

  15. Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

    OpenAIRE

    Patick, A K; Boritzki, T J; Bloom, L A

    1997-01-01

    Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritona...

  16. Profiling gene expression induced by protease-activated receptor 2 (PAR2 activation in human kidney cells.

    Directory of Open Access Journals (Sweden)

    Jacky Y Suen

    Full Text Available Protease-Activated Receptor-2 (PAR2 has been implicated through genetic knockout mice with cytokine regulation and arthritis development. Many studies have associated PAR2 with inflammatory conditions (arthritis, airways inflammation, IBD and key events in tumor progression (angiogenesis, metastasis, but they have relied heavily on the use of single agonists to identify physiological roles for PAR2. However such probes are now known not to be highly selective for PAR2, and thus precisely what PAR2 does and what mechanisms of downstream regulation are truly affected remain obscure. Effects of PAR2 activation on gene expression in Human Embryonic Kidney cells (HEK293, a commonly studied cell line in PAR2 research, were investigated here by comparing 19,000 human genes for intersecting up- or down-regulation by both trypsin (an endogenous protease that activates PAR2 and a PAR2 activating hexapeptide (2f-LIGRLO-NH(2. Among 2,500 human genes regulated similarly by both agonists, there were clear associations between PAR2 activation and cellular metabolism (1,000 genes, the cell cycle, the MAPK pathway, HDAC and sirtuin enzymes, inflammatory cytokines, and anti-complement function. PAR-2 activation up-regulated four genes more than 5 fold (DUSP6, WWOX, AREG, SERPINB2 and down-regulated another six genes more than 3 fold (TXNIP, RARG, ITGB4, CTSD, MSC and TM4SF15. Both PAR2 and PAR1 activation resulted in up-regulated expression of several genes (CD44, FOSL1, TNFRSF12A, RAB3A, COPEB, CORO1C, THBS1, SDC4 known to be important in cancer. This is the first widespread profiling of specific activation of PAR2 and provides a valuable platform for better understanding key mechanistic roles of PAR2 in human physiology. Results clearly support the development of both antagonists and agonists of human PAR2 as potential disease modifying therapeutic agents.

  17. Supermarket Proteases.

    Science.gov (United States)

    Hagar, William G.; Bullerwell, Lornie D.

    2003-01-01

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

  18. Earthworm Protease

    Directory of Open Access Journals (Sweden)

    Rong Pan

    2010-01-01

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

  19. Earthworm Protease

    International Nuclear Information System (INIS)

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

    2010-01-01

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

  20. Suboptimal inhibition of protease activity in human immunodeficiency virus type 1: Effects on virion morphogenesis and RNA maturation

    International Nuclear Information System (INIS)

    Moore, Michael D.; Fu, William; Soheilian, Ferri; Nagashima, Kunio; Ptak, Roger G.; Pathak, Vinay K.; Hu, Wei-Shau

    2008-01-01

    Protease activity within nascently released human immunodeficiency virus type 1 (HIV-1) particles is responsible for the cleavage of the viral polyproteins Gag and Gag-Pol into their constituent parts, which results in the subsequent condensation of the mature conical core surrounding the viral genomic RNA. Concomitant with viral maturation is a conformational change in the packaged viral RNA from a loosely associated dimer into a more thermodynamically stable form. In this study we used suboptimal concentrations of two protease inhibitors, lopinavir and atazanavir, to study their effects on Gag polyprotein processing and on the properties of the RNA in treated virions. Analysis of the treated virions demonstrated that even with high levels of inhibition of viral infectivity (IC 90 ), most of the Gag and Gag-Pol polyproteins were processed, although slight but significant increases in processing intermediates of Gag were detected. Drug treatments also caused a significant increase in the proportion of viruses displaying either immature or aberrant mature morphologies. The aberrant mature particles were characterized by an electron-dense region at the viral periphery and an electron-lucent core structure in the viral center, possibly indicating exclusion of the genomic RNA from these viral cores. Intriguingly, drug treatments caused only a slight decrease in overall thermodynamic stability of the viral RNA dimer, suggesting that the dimeric viral RNA was able to mature in the absence of correct core condensation

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

    DEFF Research Database (Denmark)

    Batten, MR; Senior, BW; Kilian, Mogens

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Garabaya Cecilia

    2006-03-01

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

  3. Replacement of C305 in heart/muscle-type isozyme of human carnitine palmitoyltransferase I with aspartic acid and other amino acids.

    Science.gov (United States)

    Matsuo, Taisuke; Yamamoto, Atsushi; Yamamoto, Takenori; Otsuki, Kaoru; Yamazaki, Naoshi; Kataoka, Masatoshi; Terada, Hiroshi; Shinohara, Yasuo

    2010-04-01

    Liver- and heart/muscle-type isozymes of human carnitine palmitoyltransferase I (L- and M-CPTI, respectively) show a certain similarity in their amino acid sequences, and mutation studies on the conserved amino acids between these two isozymes often show essentially the same effects on their enzymatic properties. Earlier mutation studies on C305 in human M-CPTI and its counterpart residue, C304, in human L-CPTI showed distinct effects of the mutations, especially in the aspect of enzyme stability; however, simple comparison of these effects on the conserved Cys residue between L- and M-CPTI was difficult, because these studies were carried out using different expression systems and distinct amino acids as replacements. In the present study, we carried out mutation studies on the C305 in human M-CPTI using COS cells for the expression system. Our results showed that C305 was replaceable with aspartic acid but that substitution with other amino acids caused both loss of function and reduced expression.

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

    DEFF Research Database (Denmark)

    Senior, BW; Batten, MR; Kilian, Mogens

    2002-01-01

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

  5. Cloning and chromosomal assignment of a human cDNA encoding a T cell- and natural killer cell-specific trypsin-like serine protease

    International Nuclear Information System (INIS)

    Gershenfeld, H.K.; Hershberger, R.J.; Shows, T.B.; Weissman, I.L.

    1988-01-01

    A cDNA clone encoding a human T cell- and natural killer cell-specific serine protease was obtained by screening a phage λgt10 cDNA library from phytohemagglutinin-stimulated human peripheral blood lymphocytes with the mouse Hanukah factor cDNA clone. In an RNA blot-hybridization analysis, this human Hanukah factor cDNA hybridized with a 1.3-kilobase band in allogeneic-stimulated cytotoxic T cells and the Jurkat cell line, but this transcript was not detectable in normal muscle, liver, tonsil, or thymus. By dot-blot hybridization, this cDNA hybridized with RNA from three cytolytic T-cell clones and three noncytolytic T-cell clones grown in vitro as well as with purified CD16 + natural killer cells and CD3 + , CD16 - T-cell large granular lymphocytes from peripheral blood lymphocytes (CD = cluster designation). The nucleotide sequence of this cDNA clone encodes a predicted serine protease of 262 amino acids. The active enzyme is 71% and 77% similar to the mouse sequence at the amino acid and DNA level, respectively. The human and mouse sequences conserve the active site residues of serine proteases--the trypsin-specific Asp-189 and all 10 cysteine residues. The gene for the human Hanukah factor serine protease is located on human chromosome 5. The authors propose that this trypsin-like serine protease may function as a common component necessary for lysis of target cells by cytotoxic T lymphocytes and natural killer cells

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

    Science.gov (United States)

    Antoniou, Georgia; Papakyriacou, Irineos; Papaneophytou, Christos

    2017-10-01

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

  7. β-N-Methylamino-L-alanine (BMAA) perturbs alanine, aspartate and glutamate metabolism pathways in human neuroblastoma cells as determined by metabolic profiling.

    Science.gov (United States)

    Engskog, Mikael K R; Ersson, Lisa; Haglöf, Jakob; Arvidsson, Torbjörn; Pettersson, Curt; Brittebo, Eva

    2017-05-01

    β-Methylamino-L-alanine (BMAA) is a non-proteinogenic amino acid that induces long-term cognitive deficits, as well as an increased neurodegeneration and intracellular fibril formation in the hippocampus of adult rodents following short-time neonatal exposure and in vervet monkey brain following long-term exposure. It has also been proposed to be involved in the etiology of neurodegenerative disease in humans. The aim of this study was to identify metabolic effects not related to excitotoxicity or oxidative stress in human neuroblastoma SH-SY5Y cells. The effects of BMAA (50, 250, 1000 µM) for 24 h on cells differentiated with retinoic acid were studied. Samples were analyzed using LC-MS and NMR spectroscopy to detect altered intracellular polar metabolites. The analysis performed, followed by multivariate pattern recognition techniques, revealed significant perturbations in protein biosynthesis, amino acid metabolism pathways and citrate cycle. Of specific interest were the BMAA-induced alterations in alanine, aspartate and glutamate metabolism and as well as alterations in various neurotransmitters/neuromodulators such as GABA and taurine. The results indicate that BMAA can interfere with metabolic pathways involved in neurotransmission in human neuroblastoma cells.

  8. Processing Proteases

    DEFF Research Database (Denmark)

    Ødum, Anders Sebastian Rosenkrans

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

  9. Preclinical Characterization and Human Microdose Pharmacokinetics of ITMN-8187, a Nonmacrocyclic Inhibitor of the Hepatitis C Virus NS3 Protease.

    Science.gov (United States)

    Rajagopalan, Ravi; Pan, Lin; Schaefer, Caralee; Nicholas, John; Lim, Sharlene; Misialek, Shawn; Stevens, Sarah; Hooi, Lisa; Aleskovski, Natalia; Ruhrmund, Donald; Kossen, Karl; Huang, Lea; Yap, Sophia; Beigelman, Leonid; Serebryany, Vladimir; Liu, Jyanwei; Sastry, Srikonda; Seiwert, Scott; Buckman, Brad

    2017-01-01

    The current paradigm for the treatment of chronic hepatitis C virus (HCV) infection involves combinations of agents that act directly on steps of the HCV life cycle. Here we report the preclinical characteristics of ITMN-8187, a nonmacrocyclic inhibitor of the NS3/4A HCV protease. X-ray crystallographic studies of ITMN-8187 and simeprevir binding to NS3/4A protease demonstrated good agreement between structures. Low nanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 1, 2b, 4, 5, and 6. In cell-based potency assays, half-maximal reduction of genotype 1a and 1b HCV replicon RNA was afforded by 11 and 4 nM doses of ITMN-8187, respectively. Combinations of ITMN-8187 with other directly acting antiviral agents in vitro displayed additive antiviral efficacy. A 30-mg/kg of body weight dose of ITMN-8187 administered for 4 days yielded significant viral load reductions through day 5 in a chimeric mouse model of HCV. A 3-mg/kg oral dose administered to rats, dogs, or monkeys yielded concentrations in plasma 16 h after dosing that exceeded the half-maximal effective concentration of ITMN-8187. Human microdose pharmacokinetics showed low intersubject variability and prolonged oral absorption with first-order elimination kinetics compatible with once-daily dosing. These preclinical characteristics compare favorably with those of other NS3/4A inhibitors approved for the treatment of chronic HCV infection. Copyright © 2016 American Society for Microbiology.

  10. Prokaryotic Selectivity, Anti-endotoxic Activity and Protease Stability of Diastereomeric and Enantiomeric Analogs of Human Antimicrobial Peptide LL-37

    International Nuclear Information System (INIS)

    Nan, Yong Hai; Lee, Bongju; Shin, Song Yub

    2012-01-01

    LL-37 is the only antimicrobial peptide (AMP) of the human cathelicidin family. In addition to potent antimicrobial activity, LL-37 is known to have the potential to inhibit lipolysaccharide (LPS)-induced endotoxic effects. To provide the stability to proteolytic digestion and increase prokaryotic selectivity and/or anti-endotoxic activity of two Lys/Trp-substituted 19-meric anti-microbial peptides (a4-W1 and a4-W2) designed from IG-19 (residues 13-31 of LL-37), we synthesized the diastereomeric peptides (a4-W1-D and a4-W2-D) with D-amino acid substitution at positions 3, 7, 10, 13 and 17 of a4-W1 and a4-W2, respectively and the enantiomeric peptides (a4-W1-E and a4-W2-E) composed D-amino acids. The diastereomeric peptides exhibited the best prokaryotic selectivity and effective protease stability, but no or less anti-endotoxic activity. In contrast, the enantiomeric peptides had not only prokaryotic selectivity and anti-endotoxic activity but also protease stability. Our results suggest that the hydrophobicity and α-helicity of the peptide is important for anti-endotoxic activity. In particular, the enantiomeric peptides showed potent anti-endotoxic and LPS-neutralizing activities comparable to that of LL-37. Taken together, both a4-W1-E and a4-W2-E holds promise as a template for the development of peptide antibiotics for the treatment of endotoxic shock and sepsis

  11. Prokaryotic Selectivity, Anti-endotoxic Activity and Protease Stability of Diastereomeric and Enantiomeric Analogs of Human Antimicrobial Peptide LL-37

    Energy Technology Data Exchange (ETDEWEB)

    Nan, Yong Hai; Lee, Bongju; Shin, Song Yub [Chosun Univ., Gwangju (Korea, Republic of)

    2012-09-15

    LL-37 is the only antimicrobial peptide (AMP) of the human cathelicidin family. In addition to potent antimicrobial activity, LL-37 is known to have the potential to inhibit lipolysaccharide (LPS)-induced endotoxic effects. To provide the stability to proteolytic digestion and increase prokaryotic selectivity and/or anti-endotoxic activity of two Lys/Trp-substituted 19-meric anti-microbial peptides (a4-W1 and a4-W2) designed from IG-19 (residues 13-31 of LL-37), we synthesized the diastereomeric peptides (a4-W1-D and a4-W2-D) with D-amino acid substitution at positions 3, 7, 10, 13 and 17 of a4-W1 and a4-W2, respectively and the enantiomeric peptides (a4-W1-E and a4-W2-E) composed D-amino acids. The diastereomeric peptides exhibited the best prokaryotic selectivity and effective protease stability, but no or less anti-endotoxic activity. In contrast, the enantiomeric peptides had not only prokaryotic selectivity and anti-endotoxic activity but also protease stability. Our results suggest that the hydrophobicity and α-helicity of the peptide is important for anti-endotoxic activity. In particular, the enantiomeric peptides showed potent anti-endotoxic and LPS-neutralizing activities comparable to that of LL-37. Taken together, both a4-W1-E and a4-W2-E holds promise as a template for the development of peptide antibiotics for the treatment of endotoxic shock and sepsis.

  12. Downregulation of host tryptophan-aspartate containing coat (TACO gene restricts the entry and survival of Leishmania donovani in human macrophage model

    Directory of Open Access Journals (Sweden)

    Venkateswara Reddy Gogulamudi

    2015-10-01

    Full Text Available Leishmania are obligate intracellular protozoan parasites of mammalian hosts. Promastigotes of Leishmania are internalized by macrophages and transformed into amastigotes in phagosomes, and replicate in phagolysosomes. Phagosomal maturation arrest is known to play a central role in the survival of pathogenic Leishmania within activated macrophages. Recently, tryptophan-aspartate containing coat (TACO gene has been recognized as playing a crucial role in the survival of Mycobacterium tuberculosis within human macrophages by arresting the phagosome maturation process. We postulated that a similar association of TACO gene with phagosomes would prevent the vacuole from maturation in the case of Leishmania. In this study we attempted to define the effect of TACO gene downregulation on the uptake/survival of Leishmania donovani intracellularly, by treatment with Vitamin D3/Retinoic acid (RA & Chenodeoxycholic acid (CDCA/Retinoic acid (RA combinations in human THP-1 macrophages (in vitro. Treatment with these molecules downregulated the TACO gene in macrophages, resulting in reduced parasite load and marked reduction of disease progression in L. donovani infected macrophages. Taken together, these results suggest that TACO gene downregulation may play a role in subverting macrophage machinery in establishing the L.donovani replicative niche inside the host. Our study is the first to highlight the importantrole of the TACO gene in Leishmania entry, and to identify TACO gene downregulation as potential drug target against leishmaniasis.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2007-08-01

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

  14. Validation of Simultaneous Quantitative Method of HIV Protease Inhibitors Atazanavir, Darunavir and Ritonavir in Human Plasma by UPLC-MS/MS

    Directory of Open Access Journals (Sweden)

    Tulsidas Mishra

    2014-01-01

    Full Text Available Objectives. HIV protease inhibitors are used in the treatment of patients suffering from AIDS and they act at the final stage of viral replication by interfering with the HIV protease enzyme. The paper describes a selective, sensitive, and robust method for simultaneous determination of three protease inhibitors atazanavir, darunavir and ritonavir in human plasma by ultra performance liquid chromatography-tandem mass spectrometry. Materials and Methods. The sample pretreatment consisted of solid phase extraction of analytes and their deuterated analogs as internal standards from 50 μL human plasma. Chromatographic separation of analytes was performed on Waters Acquity UPLC C18 (50 × 2.1 mm, 1.7 μm column under gradient conditions using 10 mM ammonium formate, pH 4.0, and acetonitrile as the mobile phase. Results. The method was established over a concentration range of 5.0–6000 ng/mL for atazanavir, 5.0–5000 ng/mL for darunavir and 1.0–500 ng/mL for ritonavir. Accuracy, precision, matrix effect, recovery, and stability of the analytes were evaluated as per US FDA guidelines. Conclusions. The efficiency of sample preparation, short analysis time, and high selectivity permit simultaneous estimation of these inhibitors. The validated method can be useful in determining plasma concentration of these protease inhibitors for therapeutic drug monitoring and in high throughput clinical studies.

  15. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs) in 3D Collagen Microspheres.

    Science.gov (United States)

    Han, Sejin; Li, Yuk Yin; Chan, Barbara Pui

    2016-01-01

    Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs) be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

  16. Extracellular Protease Inhibition Alters the Phenotype of Chondrogenically Differentiating Human Mesenchymal Stem Cells (MSCs in 3D Collagen Microspheres.

    Directory of Open Access Journals (Sweden)

    Sejin Han

    Full Text Available Matrix remodeling of cells is highly regulated by proteases and their inhibitors. Nevertheless, how would the chondrogenesis of mesenchymal stem cells (MSCs be affected, when the balance of the matrix remodeling is disturbed by inhibiting matrix proteases, is incompletely known. Using a previously developed collagen microencapsulation platform, we investigated whether exposing chondrogenically differentiating MSCs to intracellular and extracellular protease inhibitors will affect the extracellular matrix remodeling and hence the outcomes of chondrogenesis. Results showed that inhibition of matrix proteases particularly the extracellular ones favors the phenotype of fibrocartilage rather than hyaline cartilage in chondrogenically differentiating hMSCs by upregulating type I collagen protein deposition and type II collagen gene expression without significantly altering the hypertrophic markers at gene level. This study suggests the potential of manipulating extracellular proteases to alter the outcomes of hMSC chondrogenesis, contributing to future development of differentiation protocols for fibrocartilage tissues for intervertebral disc and meniscus tissue engineering.

  17. Podosomes, But Not the Maturation Status, Determine the Protease-Dependent 3D Migration in Human Dendritic Cells.

    Science.gov (United States)

    Cougoule, Céline; Lastrucci, Claire; Guiet, Romain; Mascarau, Rémi; Meunier, Etienne; Lugo-Villarino, Geanncarlo; Neyrolles, Olivier; Poincloux, Renaud; Maridonneau-Parini, Isabelle

    2018-01-01

    Dendritic cells (DC) are professional Antigen-Presenting Cells scattered throughout antigen-exposed tissues and draining lymph nodes, and survey the body for pathogens. Their ability to migrate through tissues, a 3D environment, is essential for an effective immune response. Upon infection, recognition of Pathogen-Associated Molecular Patterns (PAMP) by Toll-like receptors (TLR) triggers DC maturation. Mature DC (mDC) essentially use the protease-independent, ROCK-dependent amoeboid mode in vivo , or in collagen matrices in vitro . However, the mechanisms of 3D migration used by human immature DC (iDC) are still poorly characterized. Here, we reveal that human monocyte-derived DC are able to use two migration modes in 3D. In porous matrices of fibrillar collagen I, iDC adopted the amoeboid migration mode. In dense matrices of gelled collagen I or Matrigel, iDC used the protease-dependent, ROCK-independent mesenchymal migration mode. Upon TLR4 activation by LPS, mDC-LPS lose the capacity to form podosomes and degrade the matrix along with impaired mesenchymal migration. TLR2 activation by Pam 3 CSK 4 resulted in DC maturation, podosome maintenance, and efficient mesenchymal migration. Under all these conditions, when DC used the mesenchymal mode in dense matrices, they formed 3D podosomes at the tip of cell protrusions. Using PGE 2 , known to disrupt podosomes in DC, we observed that the cells remained in an immature status and the mesenchymal migration mode was abolished. We also observed that, while CCL5 (attractant of iDC) enhanced both amoeboid and mesenchymal migration of iDC, CCL19 and CCL21 (attractants of mDC) only enhanced mDC-LPS amoeboid migration without triggering mesenchymal migration. Finally, we examined the migration of iDC in tumor cell spheroids, a tissue-like 3D environment. We observed that iDC infiltrated spheroids of tumor cells using both migration modes. Altogether, these results demonstrate that human DC adopt the mesenchymal mode to

  18. Small protease sensitive oligomers of PrPSc in distinct human prions determine conversion rate of PrP(C.

    Directory of Open Access Journals (Sweden)

    Chae Kim

    Full Text Available The mammalian prions replicate by converting cellular prion protein (PrP(C into pathogenic conformational isoform (PrP(Sc. Variations in prions, which cause different disease phenotypes, are referred to as strains. The mechanism of high-fidelity replication of prion strains in the absence of nucleic acid remains unsolved. We investigated the impact of different conformational characteristics of PrP(Sc on conversion of PrP(C in vitro using PrP(Sc seeds from the most frequent human prion disease worldwide, the Creutzfeldt-Jakob disease (sCJD. The conversion potency of a broad spectrum of distinct sCJD prions was governed by the level, conformation, and stability of small oligomers of the protease-sensitive (s PrP(Sc. The smallest most potent prions present in sCJD brains were composed only of∼20 monomers of PrP(Sc. The tight correlation between conversion potency of small oligomers of human sPrP(Sc observed in vitro and duration of the disease suggests that sPrP(Sc conformers are an important determinant of prion strain characteristics that control the progression rate of the disease.

  19. Aspartate aminotransferase (AST) blood test

    Science.gov (United States)

    ... gov/ency/article/003472.htm Aspartate aminotransferase (AST) blood test To use the sharing features on this page, please enable JavaScript. The aspartate aminotransferase (AST) blood test measures the level of the enzyme AST in ...

  20. Increased expression of protease-activated receptor 4 and Trefoil factor 2 in human colorectal cancer.

    Directory of Open Access Journals (Sweden)

    Guoyu Yu

    Full Text Available Protease-activated receptor 4 (PAR4, a member of G-protein coupled receptors family, was recently reported to exhibit decreased expression in gastric cancer and esophageal squamous cancer, yet increased expression during the progression of prostate cancer. Trefoil factor 2 (TFF2, a small peptide constitutively expressed in the gastric mucosa, plays a protective role in restitution of gastric mucosa. Altered TFF2 expression was also related to the development of gastrointestinal cancer. TFF2 has been verified to promote cell migration via PAR4, but the roles of PAR4 and TFF2 in the progress of colorectal cancer are still unknown. In this study, the expression level of PAR4 and TFF2 in colorectal cancer tissues was measured using real-time PCR (n = 38, western blotting (n=38 and tissue microarrays (n = 66. The mRNA and protein expression levels of PAR4 and TFF2 were remarkably increased in colorectal cancer compared with matched noncancerous tissues, especially in positive lymph node and poorly differentiated cancers. The colorectal carcinoma cell LoVo showed an increased response to TFF2 as assessed by cell invasion upon PAR4 expression. However, after intervention of PAR4 expression, PAR4 positive colorectal carcinoma cell HT-29 was less responsive to TFF2 in cell invasion. Genomic bisulfite sequencing showed the hypomethylation of PAR4 promoter in colorectal cancer tissues and the hypermethylation in the normal mucosa that suggested the low methylation of promoter was correlated to the increased PAR4 expression. Taken together, the results demonstrated that the up-regulated expression of PAR4 and TFF2 frequently occurs in colorectal cancer tissues, and that overexpression of PAR4 may be resulted from promoter hypomethylation. While TFF2 promotes invasion activity of LoVo cells overexpressing PAR4, and this effect was significantly decreased when PAR4 was knockdowned in HT-29 cells. Our findings will be helpful in further investigations into the

  1. PSA-selective activation of cytotoxic human serine proteases within the tumor microenvironment as a therapeutic strategy to target prostate cancer.

    Science.gov (United States)

    Rogers, Oliver C; Anthony, Lizamma; Rosen, D Marc; Brennen, W Nathaniel; Denmeade, Samuel R

    2018-04-27

    Prostate cancer is the most diagnosed malignancy and the second leading cause of cancer-related death in American men. While localized therapy is highly curative, treatments for metastatic prostate cancer are largely palliative. Thus, new innovative therapies are needed to target metastatic tumors. Prostate-Specific Antigen (PSA) is a chymotrypsin-like protease with a unique substrate specificity that is secreted by both normal and malignant prostate epithelial cells. Previous studies demonstrated the presence of high levels (μM-mM) of enzymatically active PSA is present in the extracellular fluid of the prostate cancer microenvironment. Because of this, PSA is an attractive target for a protease activated pro-toxin therapeutic strategy. Because prostate cancers typically grow very slowly, a strategy employing a proliferation-independent cytotoxic payload is preferred. Recently, it was shown that the human protease Granzyme B (GZMB), at low micromolar concentrations in the extracellular space, can cleave an array of extracellular matrix (ECM) proteins thus perturbing cell growth, signaling, motility, and integrity. It is also well established that other human proteases such as trypsin can induce similar effects. Because both enzymes require N-terminal proteolytic activation, we propose to convert these proteins into PSA-activated cytotoxins. In this study, we examine the enzymatic and cell targeting parameters of these PSA-activated cytotoxic serine proteases. These pro-enzymes were activated robustly by PSA and induced ECM damage that led to the death of prostate cancer cells in vitro thus supporting the potential use of this strategy as means to target metastatic prostate cancers.

  2. Ex Vivo and In Vivo Imaging and Biodistribution of Aptamers Targeting the Human Matrix MetalloProtease-9 in Melanomas.

    Directory of Open Access Journals (Sweden)

    David Kryza

    Full Text Available The human Matrix MetalloProtease-9 (hMMP-9 is overexpressed in tumors where it promotes the release of cancer cells thus contributing to tumor metastasis. We raised aptamers against hMMP-9, which constitutes a validated marker of malignant tumors, in order to design probes for imaging tumors in human beings. A chemically modified RNA aptamer (F3B, fully resistant to nucleases was previously described. This compound was subsequently used for the preparation of F3B-Cy5, F3B-S-acetylmercaptoacetyltriglycine (MAG and F3B-DOTA. The binding properties of these derivatives were determined by surface plasmon resonance and electrophoretic mobility shift assay. Optical fluorescence imaging confirmed the binding to hMMP-9 in A375 melanoma bearing mice. Quantitative biodistribution studies were performed at 30 min, 1h and 2 h post injection of 99mTc-MAG-aptamer and 111In-DOTA-F3B. 99mTc radiolabeled aptamer specifically detected hMMP-9 in A375 melanoma tumors but accumulation in digestive tract was very high. Following i.v. injection of 111In-DOTA-F3B, high level of radioactivity was observed in kidneys and bladder but digestive tract uptake was very limited. Tumor uptake was significantly (student t test, p<0.05 higher for 111In-DOTA-F3B with 2.0%ID/g than for the 111In-DOTA-control oligonucleotide (0.7%ID/g with tumor to muscle ratio of 4.0. Such difference in tumor accumulation has been confirmed by ex vivo scintigraphic images performed at 1h post injection and by autoradiography, which revealed the overexpression of hMMP-9 in sections of human melanomas. These results demonstrate that F3B aptamer is of interest for detecting hMMP-9 in melanoma tumor.

  3. Glyceroneogenesis is inhibited through HIV protease inhibitor-induced inflammation in human subcutaneous but not visceral adipose tissue

    Science.gov (United States)

    Leroyer, Stéphanie; Vatier, Camille; Kadiri, Sarah; Quette, Joëlle; Chapron, Charles; Capeau, Jacqueline; Antoine, Bénédicte

    2011-01-01

    Glyceroneogenesis, a metabolic pathway that participates during lipolysis in the recycling of free fatty acids to triglycerides into adipocytes, contributes to the lipid-buffering function of adipose tissue. We investigated whether glyceroneogenesis could be affected by human immunodeficiency virus (HIV) protease inhibitors (PIs) responsible or not for dyslipidemia in HIV-infected patients. We treated explants obtained from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) depots from lean individuals. We observed that the dyslipidemic PIs nelfinavir, lopinavir and ritonavir, but not the lipid-neutral PI atazanavir, increased lipolysis and decreased glyceroneogenesis, leading to an increased release of fatty acids from SAT but not from VAT. At the same time, dyslipidemic PIs decreased the amount of perilipin and increased interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion in SAT but not in VAT. Parthenolide, an inhibitor of the NFκB pathway, counteracted PI-induced increased inflammation and decreased glyceroneogenesis. IL-6 (100 ng) inhibited the activity of phosphoenolpyruvate carboxykinase, the key enzyme of glyceroneogenesis, in SAT but not in VAT. Our data show that dyslipidemic but not lipid-neutral PIs decreased glyceroneogenesis as a consequence of PI-induced increased inflammation in SAT that could have an affect on adipocytes and/or macrophages. These results add a new link between fat inflammation and increased fatty acids release and suggest a greater sensitivity of SAT than VAT to PI-induced inflammation. PMID:21068005

  4. Quantitation of fibroblast activation protein (FAP-specific protease activity in mouse, baboon and human fluids and organs

    Directory of Open Access Journals (Sweden)

    Fiona M. Keane

    2014-01-01

    Full Text Available The protease fibroblast activation protein (FAP is a specific marker of activated mesenchymal cells in tumour stroma and fibrotic liver. A specific, reliable FAP enzyme assay has been lacking. FAP's unique and restricted cleavage of the post proline bond was exploited to generate a new specific substrate to quantify FAP enzyme activity. This sensitive assay detected no FAP activity in any tissue or fluid of FAP gene knockout mice, thus confirming assay specificity. Circulating FAP activity was ∼20- and 1.3-fold less in baboon than in mouse and human plasma, respectively. Serum and plasma contained comparable FAP activity. In mice, the highest levels of FAP activity were in uterus, pancreas, submaxillary gland and skin, whereas the lowest levels were in brain, prostate, leukocytes and testis. Baboon organs high in FAP activity included skin, epididymis, bladder, colon, adipose tissue, nerve and tongue. FAP activity was greatly elevated in tumours and associated lymph nodes and in fungal-infected skin of unhealthy baboons. FAP activity was 14- to 18-fold greater in cirrhotic than in non-diseased human liver, and circulating FAP activity was almost doubled in alcoholic cirrhosis. Parallel DPP4 measurements concorded with the literature, except for the novel finding of high DPP4 activity in bile. The new FAP enzyme assay is the first to be thoroughly characterised and shows that FAP activity is measurable in most organs and at high levels in some. This new assay is a robust tool for specific quantitation of FAP enzyme activity in both preclinical and clinical samples, particularly liver fibrosis.

  5. Inhibition of P-glycoprotein by HIV protease inhibitors increases intracellular accumulation of berberine in murine and human macrophages.

    Directory of Open Access Journals (Sweden)

    Weibin Zha

    Full Text Available HIV protease inhibitor (PI-induced inflammatory response in macrophages is a major risk factor for cardiovascular diseases. We have previously reported that berberine (BBR, a traditional herbal medicine, prevents HIV PI-induced inflammatory response through inhibiting endoplasmic reticulum (ER stress in macrophages. We also found that HIV PIs significantly increased the intracellular concentrations of BBR in macrophages. However, the underlying mechanisms of HIV PI-induced BBR accumulation are unknown. This study examined the role of P-glycoprotein (P-gp in HIV PI-mediated accumulation of BBR in macrophages.Cultured mouse RAW264.7 macrophages, human THP-1-derived macrophages, Wild type MDCK (MDCK/WT and human P-gp transfected (MDCK/P-gp cells were used in this study. The intracellular concentration of BBR was determined by HPLC. The activity of P-gp was assessed by measuring digoxin and rhodamine 123 (Rh123 efflux. The interaction between P-gp and BBR or HIV PIs was predicated by Glide docking using Schrodinger program. The results indicate that P-gp contributed to the efflux of BBR in macrophages. HIV PIs significantly increased BBR concentrations in macrophages; however, BBR did not alter cellular HIV PI concentrations. Although HIV PIs did not affect P-gp expression, P-gp transport activities were significantly inhibited in HIV PI-treated macrophages. Furthermore, the molecular docking study suggests that both HIV PIs and BBR fit the binding pocket of P-gp, and HIV PIs may compete with BBR to bind P-gp.HIV PIs increase the concentration of BBR by modulating the transport activity of P-gp in macrophages. Understanding the cellular mechanisms of potential drug-drug interactions is critical prior to applying successful combinational therapy in the clinic.

  6. Switching from biphasic human insulin to biphasic insulin aspart 30 in type 2 diabetes: results from the ASEAN subgroup of the A₁chieve study.

    Science.gov (United States)

    Hussein, Zanariah; Lim-Abrahan, Mary Anne; Jain, Anand B; Goh, Su Yen; Soewondo, Pradana

    2013-04-01

    To evaluate the safety and effectiveness of biphasic insulin aspart 30 (BIAsp 30) in ASEAN type 2 diabetes (T2D) patients switched from biphasic human insulin (BHI) in the non-interventional 24-week A₁chieve study. Indonesian, Malaysian, Filipino and Singaporean patients switched from BHI to BIAsp 30 at their physicians' discretion were included. The incidence of serious adverse drug reactions (SADRs), including major hypoglycaemia was the primary endpoint. Changes in hypoglycaemia, glycated haemoglobin A1c (HbA1c), fasting plasma glucose (FPG), postprandial plasma glucose (PPPG), lipids, body weight and systolic blood pressure were also evaluated. Quality of life (QoL) was measured using the EQ-5D questionnaire. For the 465 patients included (mean ± SD age: 56 ± 10.3 years, diabetes duration: 9.7 ± 7.1 years, baseline HbA1c: 9.4 ± 1.8%), the mean pre-study BHI dose was 0.62 ± 0.28 IU/kg and 63.4% were dosing BHI twice daily (bid). The mean baseline BIAsp 30 dose was 0.65 ± 0.27 U/kg, titrated up to 0.71 ± 0.28 U/kg over 24 weeks, and most patients continued bid dosing. No SADRs or major hypoglycaemic episodes were reported. The proportion of patients reporting overall hypoglycaemia decreased significantly from 10.8% at baseline to 3.4% at Week 24 (p ASEAN subgroup poorly controlled on BHI. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  7. Caveolin-1 influences vascular protease activity and is a potential stabilizing factor in human atherosclerotic disease.

    Directory of Open Access Journals (Sweden)

    Juan A Rodriguez-Feo

    Full Text Available Caveolin-1 (Cav-1 is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice. This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target

  8. Molecular cloning of the large subunit of the high-Ca2+-requiring form of human Ca2+-activated neutral protease

    International Nuclear Information System (INIS)

    Imajoh, Shinobu; Aoki, Kazumasa; Ohno, Shigeo; Emori, Yasufumi; Kawasaki, Hiroshi; Sugihara, Hidemitsu; Suzuki, Koichi

    1988-01-01

    A nearly full-length cDNA clone for the large subunit of high-Ca 2+ -requiring Ca 2+ -activated neutral protease (mCANP) from human tissues has been isolated. The deduced protein, determined for the first time as an mCANP, has essentially the same structural features as those revealed previously for the large subunits of the low-Ca 2+ -requiring form (μCANP). Namely, the protein, comprising 700 amino acid residues, is characterized by four domains, containing a cysteine protease like domain and a Ca 2+ -binding domain. The overall amino acid sequence similarities of the mCANP large subunit with those of human μCANP and chicken CANP are 62% and 66%, respectively. These values are slightly lower than that observed between μCANP and chicken CANP (70%). Local sequence similarities vary with the domain, 73-78% in the cysteine protease like domain and 48-65% in the Ca 2+ -binding domain. These results suggest that CANPs with different Ca 2+ sensitivities share a common evolutionary origin and that their regulatory mechanisms are similar except for the Ca 2+ concentrations required for activation

  9. The potential of P1 site alterations in peptidomimetic protease inhibitors as suggested by virtual screening and explored by the use of C-C-coupling reagents.

    Science.gov (United States)

    Weik, Steffen; Luksch, Torsten; Evers, Andreas; Böttcher, Jark; Sotriffer, Christoph A; Hasilik, Andrej; Löffler, Hans-Gerhard; Klebe, Gerhard; Rademann, Jörg

    2006-04-01

    A synthetic concept is presented that allows the construction of peptide isostere libraries through polymer-supported C-acylation reactions. A phosphorane linker reagent is used as a carbanion equivalent; by employing MSNT as a coupling reagent, the C-acylation can be conducted without racemization. Diastereoselective reduction was effected with L-selectride. The reagent linker allows the preparation of a norstatine library with full variation of the isosteric positions including the P1 side chain that addresses the protease S1 pocket. Therefore, the concept was employed to investigate the P1 site specificity of peptide isostere inhibitors systematically. The S1 pocket of several aspartic proteases including plasmepsin II and cathepsin D was modeled and docked with approximately 500 amino acid side chains. Inspired by this virtual screen, a P1 site mutation library was designed, synthesized, and screened against three aspartic proteases (plasmepsin II, HIV protease, and cathepsin D). The potency of norstatine inhibitors was found to depend strongly on the P1 substituent. Large, hydrophobic residues such as biphenyl, 4-bromophenyl, and 4-nitrophenyl enhanced the inhibitory activity (IC50) by up to 70-fold against plasmepsin II. In addition, P1 variation introduced significant selectivity, as up to 9-fold greater activity was found against plasmepsin II relative to human cathepsin D. The active P1 site residues did not fit into the crystal structure; however, molecular dynamics simulation suggested a possible alternative binding mode.

  10. Effect of lipopolysaccharide (LPS and peptidoglycan (PGN on human mast cell numbers, cytokine production, and protease composition

    Directory of Open Access Journals (Sweden)

    Wu Yalin

    2008-08-01

    Full Text Available Abstract Background Human mast cell (HuMC maturation occurs in tissues interfacing with the external environment, exposing both mast cell progenitors and mature mast cells, to bacteria and their products. It is unknown, however, whether long- or short-term exposure to bacteria-derived toll-like receptor (TLR ligands, such as lipopolysaccharide (LPS or peptidoglycan (PGN, influences HuMC biology. Results Over 6 wks of culture, LPS had minimal effect on HuMC numbers but increased CD117, tryptase and chymase expression. PGN inhibited HuMC development. For mature mast cells, LPS in the presence of rhSCF (10 ng/ml increased CD117, tryptase, chymase and carboxypeptidase expression, primarily in CD117low HuMC. LPS decreased FcεRI expression and β-hexosaminidase release; but had no effect on LTC4 and PGD2 production. PGN reduced HuMC numbers; and CD117 and tryptase expression. IL-1β and IL-6 (in addition to IL-8 and IL-12 were detected in short-term culture supernatants of LPS treated cells, and reproduced the increases in CD117, tryptase, chymase, and carboxypeptidase expression observed in the presence of LPS. Comparative studies with mouse bone marrow-derived mast cells from wild type, but not TLR4 knockout mice, showed increases in mRNA of mouse mast cell chymases MMCP-1, MMCP-2 and MMCP-4. Conclusion PGN inhibits HuMC growth, while LPS exerts its primary effects on mature HuMC by altering cytokine production and protease composition, particularly at low concentrations of SCF. These data demonstrate the ability of bacterial products to alter HuMC mediator production, granular content, and number which may be particularly relevant at mucosal sites where HuMC are exposed to these products.

  11. Preliminary study of molecular imaging of human hepatocellular carcinoma xenograft with Gd-based MR probe containing arginine-glycine-aspartic acid chelate

    International Nuclear Information System (INIS)

    Huo Tianlong; Du Xiangke; Zhang Sen; Li Xubin; Liu Xia

    2008-01-01

    Objective: To develop a Gd-based MR probe containing arginine-glycine-aspartic acid (RGD) motif to reveal integrin αvβ3 receptor-expressed tumor. Methods: Commercially available HYNIC- RGD conjugate with co-ligand EDDA was labeled with GdCl 3 , and the mixture was isolated and purified by solid phase extract (SPE) to get the entire probe Gd-EDDA-HYNIC-RGD. Human HCC cell line BEL-7402 was cultured and the cells harvested and suspended then subcutaneously inoculated into athymic nude mice for tumor growth. In vitro cell binding assay to integrin αcβ3 receptor and cell viability experiments were conducted. Then in vivo, imaging of the three arms of xenografts were performed by MR scan with a dedicated animal coil at baseline and time points of 0, 30, 60, 90 minutes and 24 hour post-intravenous injection (p. i.) via the tail vein. Three arms of nude mice then were sacrificed for histological examination to confirm the imaging results. Results: Gd-EDDA-HYNIC-RGD was successfully isolated by SPE and validity was verified on signal enhancement through in vitro and in vivo experiments. The T 1 relaxation rate of the probe is 3.31 mmol/s; It is well tolerated to living cells when the concentration of the probe is below 0.1 μmol/ml; both BEL-7402 Human Hepatocellular Carcinoma cell line and the tumor expressed αvβ3 receptor; The RGD-ligand was observed specifically binding with αvβ3 receptor in vitro; The nude mice model bearing HHCC was well established. The signal intensity (SI) at the tumor site were 2247.6±39.0 at baseline and 2820.9±35.2 at 90 min p.i. respectively, the SI at 90 min increased less than 25% of baseline, which is statistically different (t=-38.031, P 0.05); The signal to time curve for probe-administrated group is straightforward over time in the span of 0 to 90 minute p.i. while the control arms do not show such tendency. Conclusion: Gd-EDDA-HYNIC-RGD has the potential to used as an MR probe detecting integrin αvβ3 receptor-expressed tumor

  12. Noncanonical Wnt signaling promotes osteoclast differentiation and is facilitated by the human immunodeficiency virus protease inhibitor ritonavir

    International Nuclear Information System (INIS)

    Santiago, Francisco; Oguma, Junya; Brown, Anthony M.C.; Laurence, Jeffrey

    2012-01-01

    Highlights: ► First demonstration of direct role for noncanonical Wnt in osteoclast differentiation. ► Demonstration of Ryk as a Wnt5a/b receptor in inhibition of canonical Wnt signaling. ► Modulation of noncanonical Wnt signaling by a clinically important drug, ritonavir. ► Establishes a mechanism for an important clinical problem: HIV-associated bone loss. -- Abstract: Wnt proteins that signal via the canonical Wnt/β-catenin pathway directly regulate osteoblast differentiation. In contrast, most studies of Wnt-related effects on osteoclasts involve indirect changes. While investigating bone mineral density loss in the setting of human immunodeficiency virus (HIV) infection and its treatment with the protease inhibitor ritonavir (RTV), we observed that RTV decreased nuclear localization of β-catenin, critical to canonical Wnt signaling, in primary human and murine osteoclast precursors. This occurred in parallel with upregulation of Wnt5a and Wnt5b transcripts. These Wnts typically stimulate noncanonical Wnt signaling, and this can antagonize the canonical Wnt pathway in many cell types, dependent upon Wnt receptor usage. We now document RTV-mediated upregulation of Wnt5a/b protein in osteoclast precursors. Recombinant Wnt5b and retrovirus-mediated expression of Wnt5a enhanced osteoclast differentiation from human and murine monocytic precursors, processes facilitated by RTV. In contrast, canonical Wnt signaling mediated by Wnt3a suppressed osteoclastogenesis. Both RTV and Wnt5b inhibited canonical, β-catenin/T cell factor-based Wnt reporter activation in osteoclast precursors. RTV- and Wnt5-induced osteoclast differentiation were dependent upon the receptor-like tyrosine kinase Ryk, suggesting that Ryk may act as a Wnt5a/b receptor in this context. This is the first demonstration of a direct role for Wnt signaling pathways and Ryk in regulation of osteoclast differentiation, and its modulation by a clinically important drug, ritonavir. These studies

  13. Interaction of Leptospira interrogans with Human Proteolytic Systems Enhances Dissemination through Endothelial Cells and Protease Levels

    Science.gov (United States)

    Vieira, Monica L.; Alvarez-Flores, Miryam P.; Kirchgatter, Karin; Romero, Eliete C.; Alves, Ivy J.; de Morais, Zenaide M.; Vasconcellos, Silvio A.; Chudzinski-Tavassi, Ana M.

    2013-01-01

    We have recently reported the ability of Leptospira to capture plasminogen (PLG) and generate plasmin (PLA) bound on the microbial surface in the presence of exogenous activators. In this work, we examined the effects of leptospiral PLG binding for active penetration through the endothelial cell barrier and activation. The results indicate that leptospires with PLG association or PLA activation have enhanced migration activity through human umbilical vein endothelial cell (HUVEC) monolayers compared with untreated bacteria. Leptospira cells coated with PLG were capable of stimulating the expression of PLG activators by HUVECs. Moreover, leptospires endowed with PLG or PLA promoted transcriptional upregulation matrix metalloprotease 9 (MMP-9). Serum samples from patients with confirmed leptospirosis showed higher levels of PLG activators and total MMP-9 than serum samples from normal (healthy) subjects. The highest level of PLG activators and total MMP-9 was detected with microscopic agglutination test (MAT)-negative serum samples, suggesting that this proteolytic activity stimulation occurs at the early stage of the disease. Furthermore, a gelatin zymography profile obtained for MMPs with serum samples from patients with leptospirosis appears to be specific to leptospiral infection because serum samples from patients with unrelated infectious diseases produced no similar degradation bands. Altogether, the data suggest that the Leptospira-associated PLG or PLA might represent a mechanism that contributes to bacterial penetration of endothelial cells through an activation cascade of events that enhances the proteolytic capability of the organism. To our knowledge, this is the first proteolytic activity associated with leptospiral pathogenesis described to date. PMID:23478319

  14. Activities of the human immunodeficiency virus type 1 (HIV-1) protease inhibitor nelfinavir mesylate in combination with reverse transcriptase and protease inhibitors against acute HIV-1 infection in vitro.

    Science.gov (United States)

    Patick, A K; Boritzki, T J; Bloom, L A

    1997-10-01

    Nelfinavir mesylate (formerly AG1343) is a potent and selective, nonpeptidic inhibitor of human immunodeficiency virus type 1 (HIV-1) protease that was discovered by protein structure-based design methodologies. We evaluated the antiviral and cytotoxic effects of two-drug combinations of nelfinavir with the clinically approved antiretroviral therapeutics zidovudine (ZDV), lamivudine (3TC), dideoxycytidine (ddC; zalcitabine), stavudine (d4T), didanosine (ddI), indinavir, saquinavir, and ritonavir and a three-drug combination of nelfinavir with ZDV and 3TC against an acute HIV-1 strain RF infection of CEM-SS cells in vitro. Quantitative assessment of drug interaction was evaluated by a universal response surface approach (W. R. Greco, G. Bravo, and J. C. Parsons, Pharm. Rev. 47:331-385, 1995) and by the method of M. N. Prichard and C. Shipman (Antiviral Res. 14:181-206, 1990). Both analytical methods yielded similar results and showed that the two-drug combinations of nelfinavir with the reverse transcriptase inhibitors ZDV, 3TC, ddI, d4T, and ddC and the three-drug combination with ZDV and 3TC resulted in additive to statistically significant synergistic interactions. In a similar manner, the combination of nelfinavir with the three protease inhibitors resulted in additive (ritonavir and saquinavir) to slightly antagonistic (indinavir) interactions. In all combinations, minimal cellular cytotoxicity was observed with any drug alone and in combination. These results suggest that administration of combinations of the appropriate doses of nelfinavir with other currently approved antiretroviral therapeutic agents in vivo may result in enhanced antiviral activity with no associated increase in cellular cytotoxicity.

  15. Multifunctional Mitochondrial AAA Proteases.

    Science.gov (United States)

    Glynn, Steven E

    2017-01-01

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

  16. Antimalarial activity of HIV-1 protease inhibitor in chromone series.

    Science.gov (United States)

    Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

    2014-12-01

    Increasing parasite resistance to nearly all available antimalarial drugs becomes a serious problem to human health and necessitates the need to continue the search for new effective drugs. Recent studies have shown that clinically utilized HIV-1 protease (HIV-1 PR) inhibitors can inhibit the in vitro and in vivo growth of Plasmodium falciparum. In this study, a series of chromone derivatives possessing HIV-1 PR inhibitory activity has been tested for antimalarial activity against P. falciparum (K1 multi-drug resistant strain). Chromone 15, the potent HIV-1 PR inhibitor (IC50=0.65μM), was found to be the most potent antimalarial compound with IC50=0.95μM while primaquine and tafenoquine showed IC50=2.41 and 1.95μM, respectively. Molecular docking study of chromone compounds against plasmepsin II, an aspartic protease enzyme important in hemoglobin degradation, revealed that chromone 15 exhibited the higher binding affinity (binding energy=-13.24kcal/mol) than the known PM II inhibitors. Thus, HIV-1 PR inhibitor in chromone series has the potential to be a new class of antimalarial agent. Copyright © 2014 Elsevier Inc. All rights reserved.

  17. Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis

    NARCIS (Netherlands)

    Ma, Y. H.; Bruin, T.; Tuzgol, S.; Wilson, B. I.; Roederer, G.; Liu, M. S.; Davignon, J.; Kastelein, J. J.; Brunzell, J. D.; Hayden, M. R.

    1992-01-01

    We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the

  18. Cytomegalovirus protease targeted prodrug development.

    Science.gov (United States)

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

    2013-04-01

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

  19. Temporal activation of anti- and pro-apoptotic factors in human gingival fibroblasts infected with the periodontal pathogen, Porphyromonas gingivalis: potential role of bacterial proteases in host signalling

    Directory of Open Access Journals (Sweden)

    Takehara Tadamichi

    2006-03-01

    Full Text Available Abstract Background Porphyromonas gingivalis is the foremost oral pathogen of adult periodontitis in humans. However, the mechanisms of bacterial invasion and the resultant destruction of the gingival tissue remain largely undefined. Results We report host-P. gingivalis interactions in primary human gingival fibroblast (HGF cells. Quantitative immunostaining revealed the need for a high multiplicity of infection for optimal infection. Early in infection (2–12 h, P. gingivalis activated the proinflammatory transcription factor NF-kappa B, partly via the PI3 kinase/AKT pathway. This was accompanied by the induction of cellular anti-apoptotic genes, including Bfl-1, Boo, Bcl-XL, Bcl2, Mcl-1, Bcl-w and Survivin. Late in infection (24–36 h the anti-apoptotic genes largely shut down and the pro-apoptotic genes, including Nip3, Hrk, Bak, Bik, Bok, Bax, Bad, Bim and Moap-1, were activated. Apoptosis was characterized by nuclear DNA degradation and activation of caspases-3, -6, -7 and -9 via the intrinsic mitochondrial pathway. Use of inhibitors revealed an anti-apoptotic function of NF-kappa B and PI3 kinase in P. gingivalis-infected HGF cells. Use of a triple protease mutant P. gingivalis lacking three major gingipains (rgpA rgpB kgp suggested a role of some or all these proteases in myriad aspects of bacteria-gingival interaction. Conclusion The pathology of the gingival fibroblast in P. gingivalis infection is affected by a temporal shift from cellular survival response to apoptosis, regulated by a number of anti- and pro-apoptotic molecules. The gingipain group of proteases affects bacteria-host interactions and may directly promote apoptosis by intracellular proteolytic activation of caspase-3.

  20. The dimer interfaces of protease and extra-protease domains influence the activation of protease and the specificity of GagPol cleavage.

    Science.gov (United States)

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

    2003-01-01

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

  1. Identification of the site of human mannan-binding lectin involved in the interaction with its partner serine proteases: the essential role of Lys55

    DEFF Research Database (Denmark)

    Teillet, F; Lacroix, M; Thiel, Steffen

    2007-01-01

    Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2. To ident......Mannan-binding lectin (MBL) is an oligomeric lectin that binds neutral carbohydrates on pathogens, forms complexes with MBL-associated serine proteases (MASP)-1, -2, and -3 and 19-kDa MBL-associated protein (MAp19), and triggers the complement lectin pathway through activation of MASP-2....... To identify the MASP binding site(s) of human MBL, point mutants targeting residues C-terminal to the hinge region were produced and tested for their interaction with the MASPs and MAp19 using surface plasmon resonance and functional assays. Mutation Lys(55)Ala abolished interaction with the MASPs and MAp19...... and prevented formation of functional MBL-MASP-2 complexes. Mutations Lys(55)Gln and Lys(55)Glu abolished binding to MASP-1 and -3 and strongly inhibited interaction with MAp19. Conversely, mutation Lys(55)Arg abolished interaction with MASP-2 and MAp19, but only weakened interaction with MASP-1 and -3...

  2. Distinct 3D Architecture and Dynamics of the Human HtrA2(Omi Protease and Its Mutated Variants.

    Directory of Open Access Journals (Sweden)

    Artur Gieldon

    Full Text Available HtrA2(Omi protease controls protein quality in mitochondria and plays a major role in apoptosis. Its HtrA2S306A mutant (with the catalytic serine routinely disabled for an X-ray study to avoid self-degradation is a homotrimer whose subunits contain the serine protease domain (PD and the regulatory PDZ domain. In the inactive state, a tight interdomain interface limits penetration of both PDZ-activating ligands and PD substrates into their respective target sites. We successfully crystalized HtrA2V226K/S306A, whose active counterpart HtrA2V226K has had higher proteolytic activity, suggesting higher propensity to opening the PD-PDZ interface than that of the wild type HtrA2. Yet, the crystal structure revealed the HtrA2V226K/S306A architecture typical of the inactive protein. To get a consistent interpretation of crystallographic data in the light of kinetic results, we employed molecular dynamics (MD. V325D inactivating mutant was used as a reference. Our simulations demonstrated that upon binding of a specific peptide ligand NH2-GWTMFWV-COOH, the PDZ domains open more dynamically in the wild type protease compared to the V226K mutant, whereas the movement is not observed in the V325D mutant. The movement relies on a PDZ vs. PD rotation which opens the PD-PDZ interface in a lid-like (budding flower-like in trimer fashion. The noncovalent hinges A and B are provided by two clusters of interfacing residues, harboring V325D and V226K in the C- and N-terminal PD barrels, respectively. The opening of the subunit interfaces progresses in a sequential manner during the 50 ns MD simulation. In the systems without the ligand only minor PDZ shifts relative to PD are observed, but the interface does not open. Further activation-associated events, e.g. PDZ-L3 positional swap seen in any active HtrA protein (vs. HtrA2, were not observed. In summary, this study provides hints on the mechanism of activation of wtHtrA2, the dynamics of the inactive HtrA2V325D

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

    International Nuclear Information System (INIS)

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

    1982-01-01

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

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

    International Nuclear Information System (INIS)

    Adachi, Motoyasu; Kuroki, Ryota

    2009-01-01

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

  5. Exogenous Thyropin from p41 Invariant Chain Diminishes Cysteine Protease Activity and Affects IL-12 Secretion during Maturation of Human Dendritic Cells.

    Directory of Open Access Journals (Sweden)

    Tina Zavašnik-Bergant

    Full Text Available Dendritic cells (DC play a pivotal role as antigen presenting cells (APC and their maturation is crucial for effectively eliciting an antigen-specific immune response. The p41 splice variant of MHC class II-associated chaperone, called invariant chain p41 Ii, contains an amino acid sequence, the p41 fragment, which is a thyropin-type inhibitor of proteolytic enzymes. The effects of exogenous p41 fragment and related thyropin inhibitors acting on human immune cells have not been reported yet. In this study we demonstrate that exogenous p41 fragment can enter the endocytic pathway of targeted human immature DC. Internalized p41 fragment has contributed to the total amount of the immunogold labelled p41 Ii-specific epitope, as quantified by transmission electron microscopy, in particular in late endocytic compartments with multivesicular morphology where antigen processing and binding to MHC II take place. In cell lysates of treated immature DC, diminished enzymatic activity of cysteine proteases has been confirmed. Internalized exogenous p41 fragment did not affect the perinuclear clustering of acidic cathepsin S-positive vesicles typical of mature DC. p41 fragment is shown to interfere with the nuclear translocation of NF-κB p65 subunit in LPS-stimulated DC. p41 fragment is also shown to reduce the secretion of interleukin-12 (IL-12/p70 during the subsequent maturation of treated DC. The inhibition of proteolytic activity of lysosomal cysteine proteases in immature DC and the diminished capability of DC to produce IL-12 upon their subsequent maturation support the immunomodulatory potential of the examined thyropin from p41 Ii.

  6. Neutrophilia, gelatinase release and microvascular leakage induced by human mast cell tryptase in a mouse model: Lack of a role of protease-activated receptor 2 (PAR2).

    Science.gov (United States)

    Khedr, M E M S; Abdelmotelb, A M; Pender, S L F; Zhou, X; Walls, A F

    2018-05-01

    Tryptase, the most abundant protease of the human mast cell, has been implicated as a key mediator of allergic inflammation that acts through activation of PAR2. To investigate the contribution of PAR2 in the pro-inflammatory actions mediated by tryptase in a mice model. We have injected recombinant human βII-tryptase into the peritoneum of PAR2-deficient and wild-type C57BL/6 mice. After 6, 12 and 24 hours, mice were killed, peritoneal lavage performed and inflammatory changes investigated. Tryptase stimulated an increase in neutrophil numbers in the peritoneum, but responses did not differ between PAR2-deficient and wild-type mice. Heat inactivation of tryptase or pre-incubation with a selective tryptase inhibitor reduced neutrophilia, but neutrophil accumulation was not elicited with a peptide agonist of PAR2 (SLIGRL-NH 2 ). Zymography indicated that tryptase stimulated the release of matrix metalloproteinases (MMP) 2 and 9 in the peritoneum of both mouse strains. Studies involving immunomagnetic isolation of neutrophils suggested that neutrophils represent the major cellular source of tryptase-induced MMP2 and MMP9. At 24 hours after tryptase injection, there was increased microvascular leakage as indicated by high levels of albumin in peritoneal lavage fluid, and this appeared to be partially abolished by heat-inactivating tryptase or addition of a protease inhibitor. There was no corresponding increase in levels of histamine or total protein. The extent of tryptase-induced microvascular leakage or gelatinase release into the peritoneum did not differ between PAR2-deficient and wild-type mice. Our findings indicate that tryptase is a potent stimulus for neutrophil accumulation, MMP release and microvascular leakage. Although these actions required an intact catalytic site, the primary mechanism of tryptase in vivo would appear to involve processes independent of PAR2. © 2018 The Authors. Clinical & Experimental Allergy Published by John Wiley & Sons Ltd.

  7. Tumor necrosis factor-α and receptor activator of nuclear factor-κB ligand augment human macrophage foam-cell destruction of extracellular matrix through protease-mediated processes

    DEFF Research Database (Denmark)

    Skjøt-Arkil, Helene; Barascuk, Natasha; Larsen, Lise

    2012-01-01

    By secreting proteases such as cathepsins and matrix metalloproteinases (MMPs), macrophage foam cells may be a major cause of ruptured atherosclerotic plaques. The aims of the present study were to investigate in vitro role of human macrophage foam cells in degrading type I collagen, a major...

  8. Repeated ketamine administration alters N-methyl-d-aspartic acid receptor subunit gene expression: Implication of genetic vulnerability for ketamine abuse and ketamine psychosis in humans

    Science.gov (United States)

    Lipsky, Robert H

    2015-01-01

    For more than 40 years following its approval by the Food and Drug Administration (FDA) as an anesthetic, ketamine, a non-competitive N-methyl-d-aspartic acid (NMDA) receptor antagonist, has been used as a tool of psychiatric research. As a psychedelic drug, ketamine induces psychotic symptoms, cognitive impairment, and mood elevation, which resemble some symptoms of schizophrenia. Recreational use of ketamine has been increasing in recent years. However, little is known of the underlying molecular mechanisms responsible for ketamine-associated psychosis. Recent animal studies have shown that repeated ketamine administration significantly increases NMDA receptor subunit gene expression, in particular subunit 1 (NR1 or GluN1) levels. This results in neurodegeneration, supporting a potential mechanism where up-regulation of NMDA receptors could produce cognitive deficits in chronic ketamine abuse patients. In other studies, NMDA receptor gene variants are associated with addictive behavior. Here, we focus on the roles of NMDA receptor gene subunits in ketamine abuse and ketamine psychosis and propose that full sequencing of NMDA receptor genes may help explain individual vulnerability to ketamine abuse and ketamine-associated psychosis. PMID:25245072

  9. Repeated ketamine administration alters N-methyl-D-aspartic acid receptor subunit gene expression: implication of genetic vulnerability for ketamine abuse and ketamine psychosis in humans.

    Science.gov (United States)

    Xu, Ke; Lipsky, Robert H

    2015-02-01

    For more than 40 years following its approval by the Food and Drug Administration (FDA) as an anesthetic, ketamine, a non-competitive N-methyl-D-aspartic acid (NMDA) receptor antagonist, has been used as a tool of psychiatric research. As a psychedelic drug, ketamine induces psychotic symptoms, cognitive impairment, and mood elevation, which resemble some symptoms of schizophrenia. Recreational use of ketamine has been increasing in recent years. However, little is known of the underlying molecular mechanisms responsible for ketamine-associated psychosis. Recent animal studies have shown that repeated ketamine administration significantly increases NMDA receptor subunit gene expression, in particular subunit 1 (NR1 or GluN1) levels. This results in neurodegeneration, supporting a potential mechanism where up-regulation of NMDA receptors could produce cognitive deficits in chronic ketamine abuse patients. In other studies, NMDA receptor gene variants are associated with addictive behavior. Here, we focus on the roles of NMDA receptor gene subunits in ketamine abuse and ketamine psychosis and propose that full sequencing of NMDA receptor genes may help explain individual vulnerability to ketamine abuse and ketamine-associated psychosis. © 2014 by the Society for Experimental Biology and Medicine.

  10. Salmon and human thrombin differentially regulate radicular pain, glial-induced inflammation and spinal neuronal excitability through protease-activated receptor-1.

    Directory of Open Access Journals (Sweden)

    Jenell R Smith

    Full Text Available Chronic neck pain is a major problem with common causes including disc herniation and spondylosis that compress the spinal nerve roots. Cervical nerve root compression in the rat produces sustained behavioral hypersensitivity, due in part to the early upregulation of pro-inflammatory cytokines, the sustained hyperexcitability of neurons in the spinal cord and degeneration in the injured nerve root. Through its activation of the protease-activated receptor-1 (PAR1, mammalian thrombin can enhance pain and inflammation; yet at lower concentrations it is also capable of transiently attenuating pain which suggests that PAR1 activation rate may affect pain maintenance. Interestingly, salmon-derived fibrin, which contains salmon thrombin, attenuates nerve root-induced pain and inflammation, but the mechanisms of action leading to its analgesia are unknown. This study evaluates the effects of salmon thrombin on nerve root-mediated pain, axonal degeneration in the root, spinal neuronal hyperexcitability and inflammation compared to its human counterpart in the context of their enzymatic capabilities towards coagulation substrates and PAR1. Salmon thrombin significantly reduces behavioral sensitivity, preserves neuronal myelination, reduces macrophage infiltration in the injured nerve root and significantly decreases spinal neuronal hyperexcitability after painful root compression in the rat; whereas human thrombin has no effect. Unlike salmon thrombin, human thrombin upregulates the transcription of IL-1β and TNF-α and the secretion of IL-6 by cortical cultures. Salmon and human thrombins cleave human fibrinogen-derived peptides and form clots with fibrinogen with similar enzymatic activities, but salmon thrombin retains a higher enzymatic activity towards coagulation substrates in the presence of antithrombin III and hirudin compared to human thrombin. Conversely, salmon thrombin activates a PAR1-derived peptide more weakly than human thrombin. These

  11. The role of Omi/HtrA2 protease in neonatal postasphyxial serum-induced apoptosis in human kidney proximal tubule cells

    Directory of Open Access Journals (Sweden)

    Zhang Yong

    2012-01-01

    Full Text Available Omi/HtrA2, a proapoptotic mitochondrial serine protease, is involved in both caspase-dependent and caspaseindependent apoptosis. A growing body of evidence indicates that Omi/HtrA2 plays an important role in the pathogenesis of a variety of ischemia-reperfusion (I/R injuries. However, the role of Omi/HtrA2 in renal injuries that occur in neonates with asphyxia remains unknown. The present study was designed to investigate whether Omi/HtrA2 plays an important role in the types of renal injuries that are induced by neonatal postasphyxial serum. Human renal proximal tubular cell line (HK-2 cells were used as targets. A 20% serum taken from neonates one day after asphyxia was applied to target cells as an attacking factor. We initially included control and postasphyxial serum-attacked groups and later included a ucf-101 group in the study. In the postasphyxial serum-treated group, cytosolic Omi/HtrA2 and caspase-3 expression in HK-2 cells was significantly higher than in the control group. Moreover, the concentration of cytosolic caspase-3 was found to be markedly decreased in HK-2 cells in the ucf-101 group. Our results suggest both that postasphyxial serum has a potent apoptosis-inducing effect on HK-2 cells and that this effect can be partially blocked by ucf-101. Taken together, our results demonstrate for the first time that postasphyxial serum from neonates results in Omi/HtrA2 translocation from the mitochondria to the cytosol, where it promotes HK-2 cell apoptosis via a protease activity-dependent, caspase-mediated pathway.

  12. A Molecular Approach to Nested RT-PCR Using a New Set of Primers for the Detection of the Human Immunodeficiency Virus Protease Gene.

    Science.gov (United States)

    Zarei, Mohammad; Ravanshad, Mehrdad; Bagban, Ashraf; Fallahi, Shahab

    2016-07-01

    The human immunodeficiency virus (HIV-1) is the etiologic agent of AIDS. The disease can be transmitted via blood in the window period prior to the development of antibodies to the disease. Thus, an appropriate method for the detection of HIV-1 during this window period is very important. This descriptive study proposes a sensitive, efficient, inexpensive, and easy method to detect HIV-1. In this study 25 serum samples of patients under treatment and also 10 positive and 10 negative control samples were studied. Twenty-five blood samples were obtained from HIV-1-infected individuals who were receiving treatment at the acquired immune deficiency syndrome (AIDS) research center of Imam Khomeini hospital in Tehran. The identification of HIV-1-positive samples was done by using reverse transcription to produce copy deoxyribonucleic acid (cDNA) and then optimizing the nested polymerase chain reaction (PCR) method. Two pairs of primers were then designed specifically for the protease gene fragment of the nested real time-PCR (RT-PCR) samples. Electrophoresis was used to examine the PCR products. The results were analyzed using statistical tests, including Fisher's exact test, and SPSS17 software. The 325 bp band of the protease gene was observed in all the positive control samples and in none of the negative control samples. The proposed method correctly identified HIV-1 in 23 of the 25 samples. These results suggest that, in comparison with viral cultures, antibody detection by enzyme linked immunosorbent assay (ELISAs), and conventional PCR methods, the proposed method has high sensitivity and specificity for the detection of HIV-1.

  13. The crystal structure of protease Sapp1p from Candida parapsilosis in complex with the HIV protease inhibitor ritonavir

    Czech Academy of Sciences Publication Activity Database

    Dostál, Jiří; Brynda, Jiří; Hrušková-Heidingsfeldová, Olga; Pachl, Petr; Pichová, Iva; Řezáčová, Pavlína

    2012-01-01

    Roč. 27, č. 1 (2012), s. 160-165 ISSN 1475-6366 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945; GA ČR GA203/09/0820 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z50520514 Keywords : secreted aspartic protease * virulence factor * X-ray structure * candidiasis Subject RIV: CE - Biochemistry Impact factor: 1.495, year: 2012

  14. Bacterial proteases and virulence

    DEFF Research Database (Denmark)

    Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

    2013-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Sonia eVerma

    2016-04-01

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

  16. Marketed nonsteroidal anti-inflammatory agents, antihypertensives, and human immunodeficiency virus protease inhibitors: as-yet-unused weapons of the oncologists’ arsenal

    Directory of Open Access Journals (Sweden)

    Papanagnou P

    2015-05-01

    Full Text Available Panagiota Papanagnou,1 Panagiotis Baltopoulos,2 Maria Tsironi1 1Department of Nursing, Faculty of Human Movement and Quality of Life Sciences, University of Peloponnese, Sparta, 2Department of Sports Medicine and Biology of Physical Activity, Faculty of Physical Education and Sport Science, National and Kapodistrian University of Athens, Athens, Greece Abstract: Experimental data indicate that several pharmacological agents that have long been used for the management of various diseases unrelated to cancer exhibit profound in vitro and in vivo anticancer activity. This is of major clinical importance, since it would possibly aid in reassessing the therapeutic use of currently used agents for which clinicians already have experience. Further, this would obviate the time-consuming process required for the development and the approval of novel antineoplastic drugs. Herein, both pre-clinical and clinical data concerning the antineoplastic function of distinct commercially available pharmacological agents that are not currently used in the field of oncology, ie, nonsteroidal anti-inflammatory drugs, antihypertensive agents, and anti-human immunodeficiency virus agents inhibiting viral protease, are reviewed. The aim is to provide integrated information regarding not only the molecular basis of the antitumor function of these agents but also the applicability of the reevaluation of their therapeutic range in the clinical setting. Keywords: repositioning, tumorigenesis, pleiotropy, exploitation

  17. Extracellular glycosylphosphatidylinositol-anchored mannoproteins and proteases of Cryptococcus neoformans.

    Science.gov (United States)

    Eigenheer, Richard A; Jin Lee, Young; Blumwald, Eduardo; Phinney, Brett S; Gelli, Angie

    2007-06-01

    Extracellular proteins of Cryptococcus neoformans are involved in the pathogenesis of cryptococcosis, and some are immunoreactive antigens that may potentially serve as candidates for vaccine development. To further study the extracellular proteome of the human fungal pathogen Cry. neoformans, we conducted a proteomic analysis of secreted and cell wall-bound proteins with an acapsular strain of Cry. neoformans. Proteins were identified from both intact cells and cell walls. In both cases, extracellular proteins were removed with trypsin or beta-glucanase, and then all proteins/peptides were purified by solid-phase extraction, spin dialysis, and HPLC, and identified by liquid chromatography-mass spectrometry. This study identified 29 extracellular proteins with a predicted N-terminal signal sequence and also a predicted glycosylphosphatidylinositol anchor motif in more than half. Among the novel proteins identified were five glycosylphosphatidylinositol-anchored proteins with extensive Ser/Thr-rich regions but no apparent functional domains, a glycosylphosphatidylinositol-anchored aspartic protease, and a metalloprotease with structural similarity to an elastinolytic metalloprotease of Aspergillus fumigatus. This study suggests that Cry. neoformans has the machinery required to target glycosylphosphatidylinositol-anchored proteins to the cell wall, and it confirms the extracellular proteolytic ability of Cry. neoformans.

  18. Cystatins - Extra- and intracellular cysteine protease inhibitors: High-level secretion and uptake of cystatin C in human neuroblastoma cells

    DEFF Research Database (Denmark)

    Wallin, Hanna; Bjarnadottir, Maria; Vogel, Lotte

    2010-01-01

    signal peptides) for cellular export following translation. Results indicating existence of systems for significant internalisation of type 2 cystatins from the extracellular to intracellular compartments are reviewed. Data showing that human neuroblastoma cell lines generally secrete high levels...

  19. Vaccination with recombinant aspartic hemoglobinase reduces parasite load and blood loss after hookworm infection in dogs.

    Directory of Open Access Journals (Sweden)

    Alex Loukas

    2005-10-01

    Full Text Available Hookworms infect 730 million people in developing countries where they are a leading cause of intestinal blood loss and iron-deficiency anemia. At the site of attachment to the host, adult hookworms ingest blood and lyse the erythrocytes to release hemoglobin. The parasites subsequently digest hemoglobin in their intestines using a cascade of proteolysis that begins with the Ancylostoma caninum aspartic protease 1, APR-1.We show that vaccination of dogs with recombinant Ac-APR-1 induced antibody and cellular responses and resulted in significantly reduced hookworm burdens (p = 0.056 and fecal egg counts (p = 0.018 in vaccinated dogs compared to control dogs after challenge with infective larvae of A. caninum. Most importantly, vaccinated dogs were protected against blood loss (p = 0.049 and most did not develop anemia, the major pathologic sequela of hookworm disease. IgG from vaccinated animals decreased the catalytic activity of the recombinant enzyme in vitro and the antibody bound in situ to the intestines of worms recovered from vaccinated dogs, implying that the vaccine interferes with the parasite's ability to digest blood.To the best of our knowledge, this is the first report of a recombinant vaccine from a hematophagous parasite that significantly reduces both parasite load and blood loss, and it supports the development of APR-1 as a human hookworm vaccine.

  20. Cysteine Protease Zymography: Brief Review.

    Science.gov (United States)

    Wilkesman, Jeff

    2017-01-01

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

  1. Long-term clinical outcome of human immunodeficiency virus-infected patients with discordant immunologic and virologic responses to a protease inhibitor-containing regimen.

    Science.gov (United States)

    Piketty, C; Weiss, L; Thomas, F; Mohamed, A S; Belec, L; Kazatchkine, M D

    2001-05-01

    Within a prospective cohort of 150 human immunodeficiency virus (HIV)-infected patients who began first-line protease inhibitor therapy in 1996, the outcome of 42 patients with discrepant virologic and immunologic responses to antiretroviral treatment at 12 months was analyzed at 30 months of treatment. The incidence of AIDS-defining events and deaths (14%) in the group of patients with immunologic responses in the absence of a virologic response was higher than that in full-responder patients (2%); yet, the incidence in this group was lower than that in patients with no immunologic response, despite a virologic response (21%), and was lower than that in patients without an immunologic or virologic response (67%; P<.0001, log-rank test). Differences in outcome were significant (relative risk, 6.9; 95% confidence interval, 1.9-39.3) when factors for progression were compared with those of responder patients. The results support the relevance of the CD4 cell marker over plasma HIV load for predicting clinical outcome in patients who do not achieve full immunologic and virologic responses.

  2. Protease-sensitive synthetic prions.

    Directory of Open Access Journals (Sweden)

    David W Colby

    2010-01-01

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

  3. 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.

  4. Prediction of extracellular proteases of the human pathogen Helicobacter pylori reveals proteolytic activity of the Hp1018/19 protein HtrA.

    Directory of Open Access Journals (Sweden)

    Martin Löwer

    Full Text Available Exported proteases of Helicobacter pylori (H. pylori are potentially involved in pathogen-associated disorders leading to gastric inflammation and neoplasia. By comprehensive sequence screening of the H. pylori proteome for predicted secreted proteases, we retrieved several candidate genes. We detected caseinolytic activities of several such proteases, which are released independently from the H. pylori type IV secretion system encoded by the cag pathogenicity island (cagPAI. Among these, we found the predicted serine protease HtrA (Hp1019, which was previously identified in the bacterial secretome of H. pylori. Importantly, we further found that the H. pylori genes hp1018 and hp1019 represent a single gene likely coding for an exported protein. Here, we directly verified proteolytic activity of HtrA in vitro and identified the HtrA protease in zymograms by mass spectrometry. Overexpressed and purified HtrA exhibited pronounced proteolytic activity, which is inactivated after mutation of Ser205 to alanine in the predicted active center of HtrA. These data demonstrate that H. pylori secretes HtrA as an active protease, which might represent a novel candidate target for therapeutic intervention strategies.

  5. Aspartate protects Lactobacillus casei against acid stress.

    Science.gov (United States)

    Wu, Chongde; Zhang, Juan; Du, Guocheng; Chen, Jian

    2013-05-01

    The aim of this study was to investigate the effect of aspartate on the acid tolerance of L. casei. Acid stress induced the accumulation of intracellular aspartate in L. casei, and the acid-resistant mutant exhibited 32.5 % higher amount of aspartate than that of the parental strain at pH 4.3. Exogenous aspartate improved the growth performance and acid tolerance of Lactobacillus casei during acid stress. When cultivated in the presence of 50 mM aspartate, the biomass of cells increased 65.8 % compared with the control (without aspartate addition). In addition, cells grown at pH 4.3 with aspartate addition were challenged at pH 3.3 for 3 h, and the survival rate increased 42.26-fold. Analysis of the physiological data showed that the aspartate-supplemented cells exhibited higher intracellular pH (pHi), intracellular NH4 (+) content, H(+)-ATPase activity, and intracellular ATP pool. In addition, higher contents of intermediates involved in glycolysis and tricarboxylic acid cycle were observed in cells in the presence of aspartate. The increased contents of many amino acids including aspartate, arginine, leucine, isoleucine, and valine in aspartate-added cells may contribute to the regulation of pHi. Transcriptional analysis showed that the expression of argG and argH increased during acid stress, and the addition of aspartate induced 1.46- and 3.06-fold higher expressions of argG and argH, respectively, compared with the control. Results presented in this manuscript suggested that aspartate may protect L. casei against acid stress, and it may be used as a potential protectant during the production of probiotics.

  6. 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

  7. 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.

  8. Immunological changes in human immunodeficiency virus (HIV)-infected individuals during HIV-specific protease inhibitor treatment

    DEFF Research Database (Denmark)

    Ullum, H; Katzenstein, T; Aladdin, H

    1999-01-01

    The present study examines the influence of effective anti-retroviral treatment on immune function, evaluated by a broad array of immunological tests. We followed 12 individuals infected with human immunodeficiency virus (HIV) for 6 months after initiation of combination anti-retroviral treatment...

  9. Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

    Science.gov (United States)

    Tripathi, Trivendra; Abdi, Mahshid; Alizadeh, Hassan

    2014-05-29

    Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist

  10. Vibrio cholerae cytolysin causes an inflammatory response in human intestinal epithelial cells that is modulated by the PrtV protease.

    Directory of Open Access Journals (Sweden)

    Gangwei Ou

    Full Text Available BACKGROUND: Vibrio cholerae is the causal intestinal pathogen of the diarrheal disease cholera. It secretes the protease PrtV, which protects the bacterium from invertebrate predators but reduces the ability of Vibrio-secreted factor(s to induce interleukin-8 (IL-8 production by human intestinal epithelial cells. The aim was to identify the secreted component(s of V. cholerae that induces an epithelial inflammatory response and to define whether it is a substrate for PrtV. METHODOLOGY/PRINCIPAL FINDINGS: Culture supernatants of wild type V. cholerae O1 strain C6706, its derivatives and pure V. cholerae cytolysin (VCC were analyzed for the capacity to induce changes in cytokine mRNA expression levels, IL-8 and tumor necrosis factor-alpha (TNF-alpha secretion, permeability and cell viability when added to the apical side of polarized tight monolayer T84 cells used as an in vitro model for human intestinal epithelium. Culture supernatants were also analyzed for hemolytic activity and for the presence of PrtV and VCC by immunoblot analysis. CONCLUSIONS/SIGNIFICANCE: We suggest that VCC is capable of causing an inflammatory response characterized by increased permeability and production of IL-8 and TNF-alpha in tight monolayers. Pure VCC at a concentration of 160 ng/ml caused an inflammatory response that reached the magnitude of that caused by Vibrio-secreted factors, while higher concentrations caused epithelial cell death. The inflammatory response was totally abolished by treatment with PrtV. The findings suggest that low doses of VCC initiate a local immune defense reaction while high doses lead to intestinal epithelial lesions. Furthermore, VCC is indeed a substrate for PrtV and PrtV seems to execute an environment-dependent modulation of the activity of VCC that may be the cause of V. cholerae reactogenicity.

  11. Aspartic acid incorporated monolithic columns for affinity glycoprotein purification.

    Science.gov (United States)

    Armutcu, Canan; Bereli, Nilay; Bayram, Engin; Uzun, Lokman; Say, Rıdvan; Denizli, Adil

    2014-02-01

    Novel aspartic acid incorporated monolithic columns were prepared to efficiently affinity purify immunoglobulin G (IgG) from human plasma. The monolithic columns were synthesised in a stainless steel HPLC column (20 cm × 5 mm id) by in situ bulk polymerisation of N-methacryloyl-L-aspartic acid (MAAsp), a polymerisable derivative of L-aspartic acid, and 2-hydroxyethyl methacrylate (HEMA). Monolithic columns [poly(2-hydroxyethyl methacrylate-N-methacryloyl-L-aspartic acid) (PHEMAsp)] were characterised by swelling studies, Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The monolithic columns were used for IgG adsorption/desorption from aqueous solutions and human plasma. The IgG adsorption depended on the buffer type, and the maximum IgG adsorption from aqueous solution in phosphate buffer was 0.085 mg/g at pH 6.0. The monolithic columns allowed for one-step IgG purification with a negligible capacity decrease after ten adsorption-desorption cycles. Copyright © 2013 Elsevier B.V. All rights reserved.

  12. Identification and Structural Characterization of I84C and I84A Mutations That Are Associated with High-Level Resistance to Human Immunodeficiency Virus Protease Inhibitors and Impair Viral Replication▿

    Science.gov (United States)

    Mo, Hongmei; Parkin, Neil; Stewart, Kent D.; Lu, Liangjun; Dekhtyar, Tatyana; Kempf, Dale J.; Molla, Akhteruzzaman

    2007-01-01

    Two novel human immunodeficiency virus protease mutations, I84C and I84A, were identified in patient isolates. The mutants with I84C displayed high-level resistance (median, at least 56-fold) to nelfinavir and saquinavir, but the majority remained susceptible to lopinavir. In contrast, isolates with the I84A mutation exhibited ≥33-fold median increased levels of resistance to nelfinavir, indinavir, amprenavir, ritonavir, lopinavir, saquinavir, and atazanavir. Isolates with the I84A or I84C mutation tended to be more resistant than the isolates with the I84V mutation. Modeling of the structure of the mutant proteases indicated that the I84V, I84C, and I84A mutations all create unoccupied volume in the active site, with I84A introducing the greatest change in the accessible surface area from that of the wild-type structure. PMID:17101675

  13. Differential radioprotection of bone marrow and tumour cells by zinc aspartate

    International Nuclear Information System (INIS)

    Floersheim, G.L.; Chiodetti, N.; Bieri, A.

    1988-01-01

    The radioprotector zinc aspartate did not inhibit the radiotherapeutic effect of γ rays on human tumours grown as xenografts in immunosuppressed mice, while aminothiol radioprotectors afforded a slight inhibition. On the other hand, zinc aspartate significantly reduced the fall in the haematocrit and numbers of thrombocytes, erythrocytes and leucocytes caused by irradiation, indicating a sparing effect on bone marrow precursors of peripheral blood cells. This differential protection of neoplastic and normal cells may be of considerable benefit in clinical cancer radiotherapy, provided that zinc aspartate is better tolerated and has a more favourable therapeutic index in humans than aminothiol radioprotectors. (author)

  14. Skip Regulates TGF-β1-Induced Extracellular Matrix Degrading Proteases Expression in Human PC-3 Prostate Cancer Cells

    Directory of Open Access Journals (Sweden)

    Victor Villar

    2013-01-01

    Full Text Available Purpose. To determine whether Ski-interacting protein (SKIP regulates TGF-β1-stimulated expression of urokinase-type plasminogen activator (uPA, matrix metalloproteinase-9 (MMP-9, and uPA Inhibitor (PAI-1 in the androgen-independent human prostate cancer cell model. Materials and Methods. PC-3 prostate cancer cell line was used. The role of SKIP was evaluated using synthetic small interference RNA (siRNA compounds. The expression of uPA, MMP-9, and PAI-1 was evaluated by zymography assays, RT-PCR, and promoter transactivation analysis. Results. In PC-3 cells TGF-β1 treatment stimulated uPA, PAI-1, and MMP-9 expressions. The knockdown of SKIP in PC-3 cells enhanced the basal level of uPA, and TGF-β1 treatment inhibited uPA production. Both PAI-1 and MMP-9 production levels were increased in response to TGF-β1. The ectopic expression of SKIP inhibited both TGF-β1-induced uPA and MMP-9 promoter transactivation, while PAI-1 promoter response to the factor was unaffected. Conclusions. SKIP regulates the expression of uPA, PAI-1, and MMP-9 stimulated by TGF-β1 in PC-3 cells. Thus, SKIP is implicated in the regulation of extracellular matrix degradation and can therefore be suggested as a novel therapeutic target in prostate cancer treatment.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-06-30

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

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

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

    Science.gov (United States)

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

    2014-07-01

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

  18. Insulin Aspart in the Management of Diabetes Mellitus: 15 Years of Clinical Experience

    OpenAIRE

    Hermansen, Kjeld; Bohl, Mette; Schioldan, Anne Grethe

    2015-01-01

    Limiting excessive postprandial glucose excursions is an important component of good overall glycemic control in diabetes mellitus. Pharmacokinetic studies have shown that insulin aspart, which is structurally identical to regular human insulin except for the replacement of a single proline amino acid with an aspartic acid residue, has a more physiologic time?action profile (i.e., reaches a higher peak and reaches that peak sooner) than regular human insulin. As expected with this improved ph...

  19. Tunable protease-activatable virus nanonodes.

    Science.gov (United States)

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

    2014-05-27

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

  20. 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.

  1. Immunoglobulins in nasal secretions of healthy humans: structural integrity of secretory immunoglobulin A1 (IgA1) and occurrence of neutralizing antibodies to IgA1 proteases of nasal bacteria

    DEFF Research Database (Denmark)

    Kirkeby, L; Rasmussen, TT; Reinholdt, Jesper

    2000-01-01

    Certain bacteria, including overt pathogens as well as commensals, produce immunoglobulin A1 (IgA1) proteases. By cleaving IgA1, including secretory IgA1, in the hinge region, these enzymes may interfere with the barrier functions of mucosal IgA antibodies, as indicated by experiments in vitro....... Previous studies have suggested that cleavage of IgA1 in nasal secretions may be associated with the development and perpetuation of atopic disease. To clarify the potential effect of IgA1 protease-producing bacteria in the nasal cavity, we have analyzed immunoglobulin isotypes in nasal secretions of 11...... healthy humans, with a focus on IgA, and at the same time have characterized and quantified IgA1 protease-producing bacteria in the nasal flora of the subjects. Samples in the form of nasal wash were collected by using a washing liquid that contained lithium as an internal reference. Dilution factors and...

  2. Three-dimensional hybrid networks based on aspartic acid

    Indian Academy of Sciences (India)

    WINTEC

    Keywords. Aspartic acid; hybrid compounds; nickel aspartate; lead aspartate; achiral frameworks. ..... and coordinated to water molecules as well as car- .... (b) Dan M 2004 J. Mol. Struct. ... Sheldrick G M 1994 SADABS: Siemens area detector.

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

    Science.gov (United States)

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

    2009-01-01

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

  4. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  5. Nutritional value and proteases of Lentinus citrinus produced by solid state fermentation of lignocellulosic waste from tropical region

    Directory of Open Access Journals (Sweden)

    Ana Rita Gaia Machado

    2016-09-01

    Full Text Available This paper examined the growth and yield performance of Lentinus citrinus on cupuaçu exocarp (Theobroma grandiflorum mixed with litter (CE + LI or rice bran (Oryza sativa (CE + RB in the ratio of 2:1 (800 g:200 g to investigate the nutritional composition and proteolytic potential of the fruiting body produced. Significance values of yield were determined on substrate combinations. In CE + LI the biological efficiency of the mushrooms was 93.5% and the content of fat (4.5%, fiber (11.0%, protein (27.0% and amino acids were higher when compared with CE + RB. Among the amino acids, the amount of glutamic acid, aspartic acid, alanine, arginine and leucine was high. The biological efficiency on CE + RB reduced to 84.2% and based on the nutritional value, carbohydrates (53.59%, energy (324.33 kcal and minerals such as zinc, iron, copper, potassium and phosphorus were higher in this substrate combination. Protease activity from fruiting body was significant in CE + LI (463.55 U/mL. This protease showed an optimal activity at 50 °C in neutral and alkaline pH with maximum stability at 30 °C at alkaline pH. This is the first report of L. citrinus fruiting body nutritional composition with potential for human food and application in industrial processes.

  6. A randomized trial of insulin aspart with intensified basal NPH insulin supplementation in people with Type 1 diabetes

    NARCIS (Netherlands)

    DeVries, J. H.; Lindholm, A.; Jacobsen, J. L.; Heine, R. J.; Home, P. D.

    2003-01-01

    Aims Insulin aspart has been shown to improve post-prandial and overall glycaemic control in people with Type 1 diabetes. We hypothesized that insulin aspart with intensified basal NPH insulin supplementation would result in better overall glycaemic control than human regular insulin with standard

  7. Short hydrogen bonds in the catalytic mechanism of serine proteases

    Directory of Open Access Journals (Sweden)

    VLADIMIR LESKOVAC

    2008-04-01

    Full Text Available The survey of crystallographic data from the Protein Data Bank for 37 structures of trypsin and other serine proteases at a resolution of 0.78–1.28 Å revealed the presence of hydrogen bonds in the active site of the enzymes, which are formed between the catalytic histidine and aspartate residues and are on average 2.7 Å long. This is the typical bond length for normal hydrogen bonds. The geometric properties of the hydrogen bonds in the active site indicate that the H atom is not centered between the heteroatoms of the catalytic histidine and aspartate residues in the active site. Taken together, these findings exclude the possibility that short “low-barrier” hydrogen bonds are formed in the ground state structure of the active sites examined in this work. Some time ago, it was suggested by Cleland that the “low-barrier hydrogen bond” hypothesis is operative in the catalytic mechanism of serine proteases, and requires the presence of short hydrogen bonds around 2.4 Å long in the active site, with the H atom centered between the catalytic heteroatoms. The conclusions drawn from this work do not exclude the validity of the “low-barrier hydrogen bond” hypothesis at all, but they merely do not support it in this particular case, with this particular class of enzymes.

  8. Quantitative proteomics screen identifies a substrate repertoire of rhomboid protease RHBDL2 in human cells and implicates it in epithelial homeostasis

    Czech Academy of Sciences Publication Activity Database

    Johnson, Nicholas; Březinová, Jana; Stephens, E.; Burbridge, E.; Freeman, M.; Adrain, C.; Stříšovský, Kvido

    2017-01-01

    Roč. 7, Aug 4 (2017), č. článku 7283. ISSN 2045-2322 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 EU Projects: European Commission(XE) 304154 - Rhomboid substrates Grant - others:EMBO(DE) 2329 Institutional support: RVO:61388963 Keywords : growth factor activation * intramembrane proteases * sprouting angiogenesis Subject RIV: CE - Biochemistry OBOR OECD: Biochemistry and molecular biology Impact factor: 4.259, year: 2016 https://www.nature.com/articles/s41598-017-07556-3

  9. Cleavage of a recombinant human immunoglobulin A2 (IgA2)-IgA1 hybrid antibody by certain bacterial IgA1 proteases

    DEFF Research Database (Denmark)

    Senior, B; Dunlop, JI; Batten, MR

    2000-01-01

    , Streptococcus pneumoniae, S. sanguis, Neisseria meningitidis types 1 and 2, N. gonorrhoeae types 1 and 2, and Haemophilus influenzae type 2. Thus, for these enzymes the recognition site for IgA1 cleavage is contained within half of the IgA1 hinge region; additional distal elements, if required, are provided...... by either an IgA1 or an IgA2 framework. In contrast, the IgA2/A1 hybrid appeared to be resistant to cleavage with S. oralis and some H. influenzae type 1 IgA1 proteases, suggesting these enzymes require additional determinants for efficient substrate recognition....

  10. Processing sites in the human immunodeficiency virus type 1 (HIV-1 Gag-Pro-Pol precursor are cleaved by the viral protease at different rates

    Directory of Open Access Journals (Sweden)

    Lindquist Jeffrey N

    2005-11-01

    Full Text Available Abstract We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

  11. Processing sites in the human immunodeficiency virus type 1 (HIV-1) Gag-Pro-Pol precursor are cleaved by the viral protease at different rates.

    Science.gov (United States)

    Pettit, Steve C; Lindquist, Jeffrey N; Kaplan, Andrew H; Swanstrom, Ronald

    2005-11-01

    We have examined the kinetics of processing of the HIV-1 Gag-Pro-Pol precursor in an in vitro assay with mature protease added in trans. The processing sites were cleaved at different rates to produce distinct intermediates. The initial cleavage occurred at the p2/NC site. Intermediate cleavages occurred at similar rates at the MA/CA and RT/IN sites, and to a lesser extent at sites upstream of RT. Late cleavages occurred at the sites flanking the protease (PR) domain, suggesting sequestering of these sites. We observed paired intermediates indicative of half- cleavage of RT/RH site, suggesting that the RT domain in Gag-Pro-Pol was in a dimeric form under these assay conditions. These results clarify our understanding of the processing kinetics of the Gag-Pro-Pol precursor and suggest regulated cleavage. Our results further suggest that early dimerization of the PR and RT domains may serve as a regulatory element to influence the kinetics of processing within the Pol domain.

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

    Directory of Open Access Journals (Sweden)

    Petrova Anna

    2007-07-01

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

  13. Nepenthesin protease activity indicates digestive fluid dynamics in carnivorous nepenthes plants.

    Directory of Open Access Journals (Sweden)

    Franziska Buch

    Full Text Available Carnivorous plants use different morphological features to attract, trap and digest prey, mainly insects. Plants from the genus Nepenthes possess specialized leaves called pitchers that function as pitfall-traps. These pitchers are filled with a digestive fluid that is generated by the plants themselves. In order to digest caught prey in their pitchers, Nepenthes plants produce various hydrolytic enzymes including aspartic proteases, nepenthesins (Nep. Knowledge about the generation and induction of these proteases is limited. Here, by employing a FRET (fluorescent resonance energy transfer-based technique that uses a synthetic fluorescent substrate an easy and rapid detection of protease activities in the digestive fluids of various Nepenthes species was feasible. Biochemical studies and the heterologously expressed Nep II from Nepenthes mirabilis proved that the proteolytic activity relied on aspartic proteases, however an acid-mediated auto-activation mechanism was necessary. Employing the FRET-based approach, the induction and dynamics of nepenthesin in the digestive pitcher fluid of various Nepenthes plants could be studied directly with insect (Drosophila melanogaster prey or plant material. Moreover, we observed that proteolytic activity was induced by the phytohormone jasmonic acid but not by salicylic acid suggesting that jasmonate-dependent signaling pathways are involved in plant carnivory.

  14. Further studies on selective radioprotection by organic zinc salts and synergism of zinc aspartate with WR 2721

    International Nuclear Information System (INIS)

    Floersheim, G.L.; Bieri, A.

    1990-01-01

    Protection of the haematocrit and thrombocytes by small doses of the aminothiol radioprotector WR 2721 was markedly improved by the concomitant administration of small doses of zinc aspartate. Zinc aspartate was the only one of the tested zinc salts not inhibiting the regression induced by radiotherapy of human tumours grown as xenografts in immunosuppressed mice. This also applied to zinc aspartate with WR 2721. A dose of zinc aspartate which afforded synergistic haematological protection did not enhance the toxicity of WR 2721. The synergism of zinc aspartate with WR 2721 and the differential radioprotection of the combined protocol may make it possible in clinical cancer radiotherapy to obtain selective radioprotection at a lower toxicity giving an improved therapeutic ratio compared with WR 2721 alone. (author)

  15. Fragment growing exploiting dynamic combinatorial chemistry of inhibitors of the aspartic protease endothiapepsin

    NARCIS (Netherlands)

    Mondal, Milon; Groothuis, Daphne E.; Hirsch, Anna K. H.

    2015-01-01

    Fragment-based drug design (FBDD) has emerged as an efficient hit-identification and/or-optimization strategy with a higher hit rate than high-throughput screening (HTS). Whereas fragment linking is more challenging, fragment growing has become the preferred fragment-optimization strategy, requiring

  16. Aspartic Acid Protease from Botrytis cinerea Removes Haze-Forming Proteins during White Winemaking

    NARCIS (Netherlands)

    Sluyter, Van S.C.; Warnock, N.I.; Schmidt, S.; Anderson, P.; Kan, van J.A.L.; Bacic, A.; Waters, E.J.

    2013-01-01

    White wines suffer from heat-induced protein hazes during transport and storage unless the proteins are removed prior to bottling. Bentonite fining is by far the most commonly used method, but it is inefficient and creates several other process challenges. An alternative to bentonite is the

  17. Aspartic Protease Nepenthesin-1 as a Tool for Digestion in Hydrogen/Deuterium Exchange Mass Spectrometry

    Czech Academy of Sciences Publication Activity Database

    Kádek, Alan; Mrázek, Hynek; Halada, Petr; Rey, M.; Schriemer, D. C.; Man, Petr

    2014-01-01

    Roč. 86, č. 9 (2014), s. 4287-4294 ISSN 0003-2700 R&D Projects: GA ČR GAP206/12/0503; GA MŠk(CZ) EE2.3.30.0003 Institutional support: RVO:61388971 Keywords : HXMS * protein structure * peptides Subject RIV: CB - Analytical Chemistry , Separation Impact factor: 5.636, year: 2014

  18. Insecticide resistance and intracellular proteases.

    Science.gov (United States)

    Wilkins, Richard M

    2017-12-01

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

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

    Science.gov (United States)

    Jongsma, Maarten A; Beekwilder, Jules

    2011-08-01

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

  20. Corruption of innate immunity by bacterial proteases.

    Science.gov (United States)

    Potempa, Jan; Pike, Robert N

    2009-01-01

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

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

    Science.gov (United States)

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

    2018-02-01

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

  2. Prolonged pharmacological inhibition of cathepsin C results in elimination of neutrophil serine proteases

    DEFF Research Database (Denmark)

    Guarino, Carla; Hamon, Yveline; Croix, Cécile

    2017-01-01

    cyclopropyl nitrile CatC inhibitor almost totally lack elastase. We confirmed the elimination of neutrophil elastase-like proteases by prolonged inhibition of CatC in a non-human primate. We also showed that neutrophils lacking elastase-like protease activities were still recruited to inflammatory sites....... These preclinical results demonstrate that the disappearance of neutrophil elastase-like proteases as observed in PLS patients can be achieved by pharmacological inhibition of bone marrow CatC. Such a transitory inhibition of CatC might thus help to rebalance the protease load during chronic inflammatory diseases...

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

    Science.gov (United States)

    Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

    2014-01-01

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

  4. [Ulysses retrotransposon aspartate proteinase (Drosophila virilis)].

    Science.gov (United States)

    Volkov, D A; Savvateeva, L V; Dergousova, N I; Rumsh, L D

    2002-01-01

    Retrotransposones are mobile genetic elements occurring in genomes of bacteria, plants or animals. Retrotransposones were found to contain nucleotide sequences encoding proteins which are homological to retroviral aspartic proteinases. Our research has been focused on Ulysses which is mobile genetic element found in Drosophila virilis. We suggested a primary structure of Ulysses proteinase using comparative analysis of amino acid sequences of retroviral proteinases and proteinases from retrotransposones. The appropriate cDNA fragment has been cloned and expressed in E. coli. The purification of recombinant protein (12 kD) has been carried out by affinity chromatography using pepstatine-agarose. The obtained protein has proteolytic activity at optimum pH 5.5 like the majority of aspartic proteinases.

  5. Review of biphasic insulin aspart in the treatment of type 1 and 2 diabetes

    Directory of Open Access Journals (Sweden)

    Nazia Raja-Khan

    2008-01-01

    Full Text Available Nazia Raja-Khan, Sarah S Warehime, Robert A GabbayDivision of Endocrinology, Diabetes, and Metabolism, Penn State Institute for Diabetes and Obesity, Pennsylvania State University College of Medicine, Hershey, Pennsylvania, USABackground: Insulin is an effective treatment for achieving glycemic control and preventing complications in patients with diabetes. In order to make insulin therapy more acceptable to patients, newer formulations of insulin have been developed, such as biphasic insulins. Biphasic insulins conveniently provide both prandial and basal insulin in a single injection. One of the most well-studied biphasic insulins is biphasic insulin aspart 70/30.Objective: Our goal was to review the current literature on the safety and efficacy of biphasic insulin aspart in type 1 and type 2 diabetes.Methods: A MEDLINE search was conducted using the terms “biphasic insulin aspart” to identify clinical studies and reviews.Results: Biphasic insulin aspart more effectively reduces post-prandial glucose compared to other biphasic insulins and basal insulins. Compared to biphasic insulin aspart, fasting glucose levels are lower with NPH, similar with glargine, and similar or lower with biphasic human insulin. Treat-to-target trials have shown that a goal HbA1c below 6.5 or 7% can be achieved with biphasic insulin aspart. The risk of hypoglycemia is similar to or less than that seen with other biphasic insulins or NPH insulin.Conclusion: Biphasic insulin aspart 70/30 is a safe and effective treatment option for patients with diabetes.Keywords: biphasic insulin aspart, insulin, diabetes

  6. Variably protease-sensitive prionopathy in the UK: a retrospective review 1991-2008

    NARCIS (Netherlands)

    Head, M.W.; Yull, H.M.; Ritchie, D.L.; Langeveld, J.P.M.; Fletcher, N.A.; Knight, R.S.; Ironside, J.W.

    2013-01-01

    Variably protease-sensitive prionopathy is a newly described human prion disease of unknown aetiology lying out with the hitherto recognized phenotypic spectrum of Creutzfeldt-Jakob disease. Two cases that conform to the variably protease-sensitive prionopathy phenotype have been identified

  7. Protease of Stenotrophomonas sp. from Indonesian fermented food: gene cloning and analysis

    Directory of Open Access Journals (Sweden)

    Frans Kurnia

    2018-02-01

    Full Text Available Screening of proteolytic and fibrinolytic bacteria from Indonesian soy bean based fermented food Oncom revealed several potential isolates. Based on 16s rDNA gene analysis, one particular isolate with the highest proteolytic and fibrinolytic activity was identified as Stenotrophomonas sp. The protease gene was amplified to generate a 1749 bp Polymerase Chain Reaction product and BLAST analysis, revealed 90% homology with gene encoding protease enzyme from Stenotrophomonas maltophilia. The putative amino acid sequence indicated a serine protease enzyme with typical amino acid aspartate, histidine and serine in the catalytic triad. The gene was translated into a pre-pro-protein consisted of cleavage site on its N terminal and Pre-Peptidase Cterminal domain. Cloning of the protease gene in pET22b with Escherichia coli BL21 DE3 as the host showed that the gene was expressed as insoluble protein fraction. This is the first report for analysis of protease gene from food origin Stenotrophomonas sp.

  8. The Impact of Human Papilloma Viruses, Matrix Metallo-Proteinases and HIV Protease Inhibitors on the Onset and Progression of Uterine Cervix Epithelial Tumors: A Review of Preclinical and Clinical Studies

    Directory of Open Access Journals (Sweden)

    Giovanni Barillari

    2018-05-01

    Full Text Available Infection of uterine cervix epithelial cells by the Human Papilloma Viruses (HPV is associated with the development of dysplastic/hyperplastic lesions, termed cervical intraepithelial neoplasia (CIN. CIN lesions may regress, persist or progress to invasive cervical carcinoma (CC, a leading cause of death worldwide. CIN is particularly frequent and aggressive in women infected by both HPV and the Human Immunodeficiency Virus (HIV, as compared to the general female population. In these individuals, however, therapeutic regimens employing HIV protease inhibitors (HIV-PI have reduced CIN incidence and/or clinical progression, shedding light on the mechanism(s of its development. This article reviews published work concerning: (i the role of HPV proteins (including HPV-E5, E6 and E7 and of matrix-metalloproteinases (MMPs in CIN evolution into invasive CC; and (ii the effect of HIV-PI on events leading to CIN progression such as basement membrane and extracellular matrix invasion by HPV-positive CIN cells and the formation of new blood vessels. Results from the reviewed literature indicate that CIN clinical progression can be monitored by evaluating the expression of MMPs and HPV proteins and they suggest the use of HIV-PI or their derivatives for the block of CIN evolution into CC in both HIV-infected and uninfected women.

  9. [Comparative cost-effectiveness analysis between darunavir/ritonavir and other protease inhibitors in treatment-naive human immunodeficiency syndrome type 1-infected patients in Spain].

    Science.gov (United States)

    Smets, Erik; Brogan, Anita J; Hill, Andrew; Adriaenssen, Ines; Sawyer, Anthony W; Domingo-Pedrol, Pere; Gostkorzewicz, Joana; Ledesma, Francisco

    2013-01-01

    GESIDA (AIDS Study Group) has proposed preferred regimens of antiretroviral treatment as initial therapy in HIV infected patients. The objective of this analysis is to compare the costs and effectiveness of darunavir/r QD and other ritonavir-boosted (/r) protease inhibitors (PIs) currently recommended in GESIDA guidelines for treatment-naïve patients. A cost-efficacy model compared the boosted PIs recommended as preferred or alternative treatment choices, each used with a nucleoside reverse transcriptase inhibitor backbone. Efficacy was measured by 48-week virological response (viral load < 50 copies/mL) adjusted by baseline viral load and CD4 cell count. To generate efficiency frontiers and cost-efficacy ratios, one-year antiretroviral therapy costs in Spain, and 48-week efficacy values were used. The model estimated that starting treatment with darunavir/r QD was the most cost-effective choice compared with the other preferred PI/r based therapies. The average cost per patient with a virological response was lower for darunavir/r QD (13,420€) than for atazanavir/r QD (14,000€), or lopinavir/r BID (13,815€). Among the preferred PI/r-based therapies, darunavir/r QD also was estimated to be the most efficient option for treatment-naïve patients. Atazanavir/r QD and lopinavir/r BID were found to be «dominated» by darunavir/r) and, consequently, were outside the efficiency frontier of PI/r-based first-line treatment. Given a fixed budget of 10 million euros for PI/r-based first-line therapy, the model estimated that darunavir/r QD would yield more responders (745) than atazanavir/r QD (714), or lopinavir/r BID (724). At the same time, darunavir/r QD would reduce the number of individuals failing treatment (150) compared with atazanavir/r QD (172) and lopinavir/r BID (286). In this model, darunavir/r QD was found to be the most cost-effective choice, among the preferred PI/r-based therapies recommended in the Spanish guidelines for treatment-naïve patients

  10. Three and six grams supplementation of d-aspartic acid in resistance trained men.

    Science.gov (United States)

    Melville, Geoffrey W; Siegler, Jason C; Marshall, Paul Wm

    2015-01-01

    Although abundant research has investigated the hormonal effects of d-aspartic acid in rat models, to date there is limited research on humans. Previous research has demonstrated increased total testosterone levels in sedentary men and no significant changes in hormonal levels in resistance trained men. It was hypothesised that a higher dosage may be required for experienced lifters, thus this study investigated the effects of two different dosages of d-aspartic acid on basal hormonal levels in resistance trained men and explored responsiveness to d-aspartic acid based on initial testosterone levels. Twenty-four males, with a minimum of two years' experience in resistance training, (age, 24.5 ± 3.2 y; training experience, 3.4 ± 1.4 y; height, 178.5 ± 6.5 cm; weight, 84.7 ± 7.2 kg; bench press 1-RM, 105.3 ± 15.2 kg) were randomised into one of three groups: 6 g.d(-1) plain flour (D0); 3 g.d(-1) of d-aspartic acid (D3); and 6 g.d(-1) of d-aspartic acid (D6). Participants performed a two-week washout period, training four days per week. This continued through the experimental period (14 days), with participants consuming the supplement in the morning. Serum was analysed for levels of testosterone, estradiol, sex hormone binding globulin, albumin and free testosterone was determined by calculation. D-aspartic acid supplementation revealed no main effect for group in: estradiol; sex-hormone-binding-globulin; and albumin. Total testosterone was significantly reduced in D6 (P = 0.03). Analysis of free testosterone showed that D6 was significantly reduced as compared to D0 (P = 0.005), but not significantly different to D3. Analysis did not reveal any significant differences between D3 and D0. No significant correlation between initial total testosterone levels and responsiveness to d-aspartic acid was observed (r = 0.10, P = 0.70). The present study demonstrated that a daily dose of six grams of d-aspartic acid decreased

  11. Further characterization of a highly attenuated Yersinia pestis CO92 mutant deleted for the genes encoding Braun lipoprotein and plasminogen activator protease in murine alveolar and primary human macrophages.

    Science.gov (United States)

    van Lier, Christina J; Tiner, Bethany L; Chauhan, Sadhana; Motin, Vladimir L; Fitts, Eric C; Huante, Matthew B; Endsley, Janice J; Ponnusamy, Duraisamy; Sha, Jian; Chopra, Ashok K

    2015-03-01

    We recently characterized the Δlpp Δpla double in-frame deletion mutant of Yersinia pestis CO92 molecularly, biologically, and immunologically. While Braun lipoprotein (Lpp) activates toll-like receptor-2 to initiate an inflammatory cascade, plasminogen activator (Pla) protease facilitates bacterial dissemination in the host. The Δlpp Δpla double mutant was highly attenuated in evoking bubonic and pneumonic plague, was rapidly cleared from mouse organs, and generated humoral and cell-mediated immune responses to provide subsequent protection to mice against a lethal challenge dose of wild-type (WT) CO92. Here, we further characterized the Δlpp Δpla double mutant in two murine macrophage cell lines as well as in primary human monocyte-derived macrophages to gauge its potential as a live-attenuated vaccine candidate. We first demonstrated that the Δpla single and the Δlpp Δpla double mutant were unable to survive efficiently in murine and human macrophages, unlike WT CO92. We observed that the levels of Pla and its associated protease activity were not affected in the Δlpp single mutant, and, likewise, deletion of the pla gene from WT CO92 did not alter Lpp levels. Further, our study revealed that both Lpp and Pla contributed to the intracellular survival of WT CO92 via different mechanisms. Importantly, the ability of the Δlpp Δpla double mutant to be phagocytized by macrophages, to stimulate production of tumor necrosis factor-α and interleukin-6, and to activate the nitric oxide killing pathways of the host cells remained unaltered when compared to the WT CO92-infected macrophages. Finally, macrophages infected with either the WT CO92 or the Δlpp Δpla double mutant were equally efficient in their uptake of zymosan particles as determined by flow cytometric analysis. Overall, our data indicated that although the Δlpp Δpla double mutant of Y. pestis CO92 was highly attenuated, it retained the ability to elicit innate and subsequent acquired immune

  12. Effect of Orthodontic Tooth Movement on Salivary Aspartate Aminotransferase Activity

    Directory of Open Access Journals (Sweden)

    Steiven Adhitya

    2013-07-01

    Full Text Available 72 1024x768 Aspartate aminotransferase is one of biological indicator in gingival crevicular fluid (CGF. Force orthodontic application could increase activity of aspartate aminotransferase in CGF. However, the increase activity of aspartate aminotransferase in saliva due to orthodontic force and its correlation between aspartate aminotransferase activity and tooth movement remains unclear. Objectives: To evaluate application orthodontic force on the aspartate aminotransferase activity in saliva based on the duration of force and finding correlation between tooth movement and aspartate aminotransferase activity. Methods: Twenty saliva samples collected before extraction of first premolar, at the time of force application for canine retraction and after force application. The canines retraction used 100 grams of interrupted force (module chain for thirty days. The collection of saliva and the measurement of tooth movement were carried out 1 day, 7 days, 14 days, 21 days, and 28 days after force application. The measurement of aspartate aminotransferase activity in saliva was done using spectrophotometer. Results: Application of orthodontic force influences the salivary aspartate aminotransferase activity (F=25.290, p=0.000. Furthermore, tooth movement correlated with aspartate aminotransferase activity (F=0.429, p=0.000. Conclusion: Aspartate aminotransferase activity could be used as tooth movement indicator that related to the duration of force application.DOI : 10.14693/jdi.v20i1.128

  13. Breakdown of the innate immune system by bacterial proteases

    NARCIS (Netherlands)

    Laarman, A.J.

    2011-01-01

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

  14. Contemporary protease inhibitors and cardiovascular risk

    DEFF Research Database (Denmark)

    Lundgren, Jens; Mocroft, Amanda; Ryom, Lene

    2018-01-01

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

  15. Chemical Tools for the Study of Intramembrane Proteases.

    Science.gov (United States)

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

    2015-11-20

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

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

    DEFF Research Database (Denmark)

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

    2011-01-01

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

  17. Biocatalytic Enantioselective Synthesis of N-Substituted Aspartic Acids by Aspartate Ammonia Lyase

    NARCIS (Netherlands)

    Weiner, Barbara; Poelarends, Gerrit J.; Janssen, Dick B.; Feringa, Ben L.

    2008-01-01

    The gene encoding aspartate ammonia lyase (aspB) from Bacillus sp. YM55-1 has been cloned and overexpressed, and the recombinant enzyme containing a C-terminal His6 tag has been purified to homogeneity and subjected to kinetic characterization. Kinetic studies have shown that the His6 tag does not

  18. Protease signaling through protease activated receptor 1 mediate nerve activation by mucosal supernatants from irritable bowel syndrome but not from ulcerative colitis patients.

    Science.gov (United States)

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

    2018-01-01

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

  19. A genomic survey of proteases in Aspergilli

    NARCIS (Netherlands)

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

    2014-01-01

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

  20. Curcumin derivatives as HIV-1 protease inhibitors

    Energy Technology Data Exchange (ETDEWEB)

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

    1993-12-31

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

  1. tolerant alkaline protease from Bacillus coagulans PSB

    African Journals Online (AJOL)

    oyaide

    2013-05-22

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

  2. Characterization and inhibition of norovirus proteases of genogroups I and II using a fluorescence resonance energy transfer assay

    International Nuclear Information System (INIS)

    Chang, Kyeong-Ok; Takahashi, Daisuke; Prakash, Om; Kim, Yunjeong

    2012-01-01

    Noroviruses are the major cause of food- or water-borne gastroenteritis outbreaks in humans. The norovirus protease that cleaves a large viral polyprotein to nonstructural proteins is essential for virus replication and an attractive target for antiviral drug development. Noroviruses show high genetic diversity with at least five genogroups, GI–GV, of which GI and GII are responsible for the majority of norovirus infections in humans. We cloned and expressed proteases of Norwalk virus (GI) and MD145 virus (GII) and characterized the enzymatic activities with fluorescence resonance energy transfer substrates. We demonstrated that the GI and GII proteases cleaved the substrates derived from the naturally occurring cleavage site in the open reading frame (ORF) 1 of G1 norovirus with similar efficiency, and that enzymatic activity of both proteases was inhibited by commercial protease inhibitors including chymostatin. The interaction of chymostatin to Norwalk virus protease was validated by nuclear magnetic resonance (NMR) spectroscopy.

  3. Immunohistochemical localisation of d-β-aspartic acid in pingueculae

    OpenAIRE

    Kaji, Y; Oshika, T; Okamoto, F; Fujii, N

    2009-01-01

    Background: D-β-Aspartic acid residues, which are biologically uncommon, have been reported to accumulate in various proteins of the living body with age. In the present study, D-β-aspartic acid-containing proteins were found to be localised in pingueculae, which represent one of the most prominent age-related ocular changes.Methods: Surgical specimens of conjunctivae with or without pingueculae were obtained from eight patients. Immunohistochemical localisation of D-β-aspartic acid-containin...

  4. The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements

    LENUS (Irish Health Repository)

    Thornton, Roibeard F

    2010-04-23

    Abstract Background The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. Results Four homologues of the streptococcal virulence factor SpeB were identified in the B. fragilis genome. These four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. Two of these protease genes are unique to B. fragilis 638R and are associated with two large genomic insertions. Gene annotation indicated that one of these insertions was a conjugative Tn-like element and the other was a prophage-like element, which was shown to be capable of excision. Homologues of the B. fragilis C10 protease genes were present in a panel of clinical isolates, and in DNA extracted from normal human faecal microbiota. Conclusions This study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota.

  5. The dissemination of C10 cysteine protease genes in Bacteroides fragilis by mobile genetic elements

    Directory of Open Access Journals (Sweden)

    Kagawa Todd F

    2010-04-01

    Full Text Available Abstract Background The C10 family of cysteine proteases includes enzymes that contribute to the virulence of bacterial pathogens, such as SpeB in Streptococcus pyogenes. The presence of homologues of cysteine protease genes in human commensal organisms has not been examined. Bacteroides fragilis is a member of the dominant Bacteroidetes phylum of the human intestinal microbiota, and is a significant opportunistic pathogen. Results Four homologues of the streptococcal virulence factor SpeB were identified in the B. fragilis genome. These four protease genes, two were directly contiguous to open reading frames predicted to encode staphostatin-like inhibitors, with which the protease genes were co-transcribed. Two of these protease genes are unique to B. fragilis 638R and are associated with two large genomic insertions. Gene annotation indicated that one of these insertions was a conjugative Tn-like element and the other was a prophage-like element, which was shown to be capable of excision. Homologues of the B. fragilis C10 protease genes were present in a panel of clinical isolates, and in DNA extracted from normal human faecal microbiota. Conclusions This study suggests a mechanism for the evolution and dissemination of an important class of protease in major members of the normal human microbiota.

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

    Science.gov (United States)

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

    2017-06-01

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

  7. Factor VII-activating protease

    DEFF Research Database (Denmark)

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

    2017-01-01

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

  8. Carbohydrase and protease supplementation increased ...

    African Journals Online (AJOL)

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

  9. Homoserine as an Aspartic Acid Precursor for Synthesis of Proteoglycan Glycopeptide Containing Aspartic Acid and Sulfated Glycan Chain

    OpenAIRE

    Yang, Weizhun; Ramadan, Sherif; Yang, Bo; Yoshida, Keisuke; Huang, Xuefei

    2016-01-01

    Among many hurdles in synthesizing proteoglycan glycopeptides, one challenge is the incorporation of aspartic acid in the peptide backbone and acid sensitive O-sulfated glycan chains. To overcome this, a new strategy was developed utilizing homoserine as an aspartic acid precursor. The conversion of homoserine to aspartic acid in the glycopeptide was successfully accomplished by late stage oxidation using 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) and bis(acetoxy)iodobenzene (BAIB). This is...

  10. Homoserine as an Aspartic Acid Precursor for Synthesis of Proteoglycan Glycopeptide Containing Aspartic Acid and a Sulfated Glycan Chain.

    Science.gov (United States)

    Yang, Weizhun; Ramadan, Sherif; Yang, Bo; Yoshida, Keisuke; Huang, Xuefei

    2016-12-02

    Among many hurdles in synthesizing proteoglycan glycopeptides, one challenge is the incorporation of aspartic acid in the peptide backbone and acid sensitive O-sulfated glycan chains. To overcome this, a new strategy was developed utilizing homoserine as an aspartic acid precursor. The conversion of homoserine to aspartic acid in the glycopeptide was successfully accomplished by late stage oxidation using (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) and bis(acetoxy)iodobenzene (BAIB). This is the first time that a glycopeptide containing aspartic acid and an O-sulfated glycan was synthesized.

  11. Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.

    Science.gov (United States)

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

    2000-10-31

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

  12. Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

    Science.gov (United States)

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

    2015-07-01

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

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

    Science.gov (United States)

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

    2013-12-01

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

  14. Hemoglobin cleavage site-specificity of the Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3.

    Directory of Open Access Journals (Sweden)

    Shoba Subramanian

    Full Text Available The Plasmodium falciparum cysteine proteases falcipain-2 and falcipain-3 degrade host hemoglobin to provide free amino acids for parasite protein synthesis. Hemoglobin hydrolysis has been described as an ordered process initiated by aspartic proteases, but cysteine protease inhibitors completely block the process, suggesting that cysteine proteases can also initiate hemoglobin hydrolysis. To characterize the specific roles of falcipains, we used three approaches. First, using random P(1 - P(4 amino acid substrate libraries, falcipain-2 and falcipain-3 demonstrated strong preference for cleavage sites with Leu at the P(2 position. Second, with overlapping peptides spanning alpha and beta globin and proteolysis-dependent (18O labeling, hydrolysis was seen at many cleavage sites. Third, with intact hemoglobin, numerous cleavage products were identified. Our results suggest that hemoglobin hydrolysis by malaria parasites is not a highly ordered process, but rather proceeds with rapid cleavage by falcipains at multiple sites. However, falcipain-2 and falcipain-3 show strong specificity for P(2 Leu in small peptide substrates, in agreement with the specificity in optimized small molecule inhibitors that was identified previously. These results are consistent with a principal role of falcipain-2 and falcipain-3 in the hydrolysis of hemoglobin by P. falciparum and with the possibility of developing small molecule inhibitors with optimized specificity as antimalarial agents.

  15. Effect of poloxamer 407 administration on the serum lipids profile, anxiety level and protease activity in the heart and liver of mice

    Science.gov (United States)

    Johnston, Thomas P.; Dubrovina, Nina I.; Kisarova, Yana A.; Zhanaeva, Svetlana Ya.; Cherkanova, Marina S.; Filjushina, Elena E.; Alexeenko, Tatyana V.; Machova, Eva; Zhukova, Natalya A.

    2013-01-01

    Chronic administration of the poloxamer 407 (P-407), a block copolymer, to elevate serum lipids in mice is a well-established mouse model of hyperlipidemia and atherosclerosis. We tested the hypothesis that the activity of several types of proteases in heart and liver tissue is changed in the early stages of atherosclerosis development. Additionally, we evaluated whether increased serum lipids would induce anxiety in mice, as determined by using a ‘plus-maze’ test. The mice were administered P-407 by intraperitoneal injection twice a week for one month. P-407 administration to mice resulted in a marked increase in total serum cholesterol, atherogenic non-HDL-cholesterol, and especially in total triglycerides, and it also increased anxiety. Morphological changes observed in P-407-treated mice included contractile type changes in cardiomyocytes and foamy macrophages in liver. A significant increase of cysteine proteases cathepsin B and cathepsin L (at 24 h) and aspartate protease cathepsin D (at both 24 h and 5 days) was determined in heart tissue following P-407 administration. However, no changes were noted in heart matrix metalloproteinase activity. The activity of cysteine and aspartate proteases was significantly increased in liver at both 24 hours and 5 days after P-407 administration. In conclusion, administration of P-407 to mice for one month resulted in increased anxiety, and more importantly, there was an increase in the activity of heart and liver proteases secondary to sustained dyslipidemia. It is suggested that heart and liver cysteine and aspartate proteases may represent potential therapeutic targets in the early stages of atherosclerosis. PMID:24170975

  16. Racemization of aspartic acid and phenylalanine in the sweetener aspartame at 100 degrees C.

    Science.gov (United States)

    Boehm, M F; Bada, J L

    1984-01-01

    The racemization half-lives (i.e., the time required to reach a D/L = 0.33) at pH 6.8 for aspartic acid and phenylalanine in the sweetener aspartame (L-aspartyl-L-phenylalanine methyl ester) were determined to be 13 and 23 hours, respectively, at 100 degrees C. Racemization at this pH does not occur in aspartame but rather in its diketopiperazine decomposition product. Our results indicate that the use of aspartame to sweeten neutral pH foods and beverages that are then heated at elevated temperature could generate D-aspartic acid and D-phenylalanine. The nutritive consequences of these D-amino acids in the human diet are not well established, and thus aspartame should probably not be used as a sweetener when the exposure of neutral pH foods and beverages to elevated temperatures is required. At pH 4, a typical pH of most foods and beverages that might be sweetened with aspartame, the half-lives are 47 hours for aspartic acid and 1200 hours for phenylalanine at 100 degrees C. Racemization at pH 4 takes place in aspartame itself. Although the racemization rates at pH 4 are slow and no appreciable racemization of aspartic acid and phenylalanine should occur during the normal use of aspartame, some food and beverage components could conceivably act as catalysts. Additional studies are required to evaluate whether the use of aspartame as a sugar substitute might not in turn result in an increased human consumption of D-aspartic acid and D-phenylalanine. PMID:6591191

  17. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

    Energy Technology Data Exchange (ETDEWEB)

    Lizarte, F.S. Neto; Tirapelli, D.P.C. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Ambrosio, S.R. [Universidade de Franca, Núcleo de Pesquisa em Ciências e Tecnologia, Franca, SP (Brazil); Tirapelli, C.R. [Universidade de São Paulo, Laboratório de Farmacologia, Departamento de Enfermagem Psiquiátrica e Ciências Humanas, Escola de Enfermagem de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Oliveira, F.M. [Universidade de São Paulo, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Novais, P.C. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Peria, F.M.; Oliveira, H.F. [Universidade de São Paulo, Departamento de Clínica Médica, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil); Carlotti, C.G. Junior; Tirapelli, L.F. [Universidade de São Paulo, Departamento de Cirurgia e Anatomia, Faculdade de Medicina de Ribeirão Preto, Ribeirão Preto, SP (Brazil)

    2013-01-11

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors.

  18. Kaurene diterpene induces apoptosis in U87 human malignant glioblastoma cells by suppression of anti-apoptotic signals and activation of cysteine proteases

    International Nuclear Information System (INIS)

    Lizarte, F.S. Neto; Tirapelli, D.P.C.; Ambrosio, S.R.; Tirapelli, C.R.; Oliveira, F.M.; Novais, P.C.; Peria, F.M.; Oliveira, H.F.; Carlotti, C.G. Junior; Tirapelli, L.F.

    2013-01-01

    Gliomas are the most common and malignant primary brain tumors in humans. Studies have shown that classes of kaurene diterpene have anti-tumor activity related to their ability to induce apoptosis. We investigated the response of the human glioblastoma cell line U87 to treatment with ent-kaur-16-en-19-oic acid (kaurenoic acid, KA). We analyzed cell survival and the induction of apoptosis using flow cytometry and annexin V staining. Additionally, the expression of anti-apoptotic (c-FLIP and miR-21) and apoptotic (Fas, caspase-3 and caspase-8) genes was analyzed by relative quantification (real-time PCR) of mRNA levels in U87 cells that were either untreated or treated with KA (30, 50, or 70 µM) for 24, 48, and 72 h. U87 cells treated with KA demonstrated reduced viability, and an increase in annexin V- and annexin V/PI-positive cells was observed. The percentage of apoptotic cells was 9% for control cells, 26% for cells submitted to 48 h of treatment with 50 µM KA, and 31% for cells submitted to 48 h of treatment with 70 µM KA. Similarly, in U87 cells treated with KA for 48 h, we observed an increase in the expression of apoptotic genes (caspase-8, -3) and a decrease in the expression of anti-apoptotic genes (miR-21 and c-FLIP). KA possesses several interesting properties and induces apoptosis through a unique mechanism. Further experiments will be necessary to determine if KA may be used as a lead compound for the development of new chemotherapeutic drugs for the treatment of primary brain tumors

  19. Hypoglycemia in type 1 diabetic pregnancy: role of preconception insulin aspart treatment in a randomized study

    DEFF Research Database (Denmark)

    Heller, Simon; Damm, Peter; Mersebach, Henriette

    2010-01-01

    OBJECTIVE A recent randomized trial compared prandial insulin aspart (IAsp) with human insulin in type 1 diabetic pregnancy. The aim of this exploratory analysis was to investigate the incidence of severe hypoglycemia during pregnancy and compare women enrolled preconception with women enrolled...... during early pregnancy. RESEARCH DESIGN AND METHODS IAsp administered immediately before each meal was compared with human insulin administered 30 min before each meal in 99 subjects (44 to IAsp and 55 to human insulin) randomly assigned preconception and in 223 subjects (113 for IAsp and 110 for human...

  20. Hypoglycemia in type 1 diabetic pregnancy: role of preconception insulin aspart treatment in a randomized study

    DEFF Research Database (Denmark)

    Heller, Simon; Damm, Peter; Mersebach, Henriette

    2010-01-01

    OBJECTIVE A recent randomized trial compared prandial insulin aspart (IAsp) with human insulin in type 1 diabetic pregnancy. The aim of this exploratory analysis was to investigate the incidence of severe hypoglycemia during pregnancy and compare women enrolled preconception with women enrolled...... during early pregnancy. RESEARCH DESIGN AND METHODS IAsp administered immediately before each meal was compared with human insulin administered 30 min before each meal in 99 subjects (44 to IAsp and 55 to human insulin) randomly assigned preconception and in 223 subjects (113 for IAsp and 110 for human...... insulin) randomly assigned in early pregnancy (...

  1. Characterization of a secreted Chlamydia protease

    DEFF Research Database (Denmark)

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

    2002-01-01

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

  2. Deferoxamine Suppresses Collagen Cleavage and Protease, Cytokine, and COL10A1 Expression and Upregulates AMPK and Krebs Cycle Genes in Human Osteoarthritic Cartilage

    Directory of Open Access Journals (Sweden)

    Elena V. Tchetina

    2016-01-01

    Full Text Available This study reports the effects of the iron chelator deferoxamine (DFO on collagen cleavage, inflammation, and chondrocyte hypertrophy in relation to energy metabolism-related gene expression in osteoarthritic (OA articular cartilage. Full-depth explants of human OA knee articular cartilage from arthroplasty were cultured with exogenous DFO (1–50 μM. Type II collagen cleavage and phospho-adenosine monophosphate-activated protein kinase (pAMPK concentrations were measured using ELISAs. Gene expression studies employed real-time PCR and included AMPK analyses in PBMCs. In OA explants collagen cleavage was frequently downregulated by 10–50 μM DFO. PCR analysis of 7 OA patient cartilages revealed that 10 μM DFO suppressed expression of MMP-1, MMP-13, IL-1β, and TNFα and a marker of chondrocyte hypertrophy, COL10A1. No changes were observed in the expression of glycolysis-related genes. In contrast, expressions of genes associated with the mitochondrial Krebs cycle (TCA, AMPK, HIF1α, and COL2A1 were upregulated. AMPK gene expression was reduced in OA cartilage and increased in PBMCs from the same patients compared to healthy controls. Our studies demonstrate that DFO is capable of suppressing excessive collagenase-mediated type II collagen cleavage in OA cartilage and reversing phenotypic changes. The concomitant upregulation of proanabolic TCA-related gene expressions points to a potential for availability of energy generating substrates required for matrix repair by end-stage OA chondrocytes. This might normally be prevented by high whole-body energy requirements indicated by elevated AMPK expression in PBMCs of OA patients.

  3. Advances in protease engineering for laundry detergents.

    Science.gov (United States)

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

    2015-12-25

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

  4. In vitro effects of sodium benzoate on the activities of aspartate and ...

    African Journals Online (AJOL)

    The in vitro effects of varying concentrations sodium benzoate on the activities of aspartate (E.C. 2.6.1.1) and alanine (E.C. 2.6.1.2) aminotransferases (AST and ALT, respectively) and alkaline phosphatase (E.C. 3.1.3.1; abbreviated as ALP) from human erythrocytes of different genotypes (HbAA, HbAS and HbSS) were ...

  5. Aspartate inhibits Staphylococcus aureus biofilm formation.

    Science.gov (United States)

    Yang, Hang; Wang, Mengyue; Yu, Junping; Wei, Hongping

    2015-04-01

    Biofilm formation renders Staphylococcus aureus highly resistant to conventional antibiotics and host defenses. Four D-amino acids (D-Leu, D-Met, D-Trp and D-Tyr) have been reported to be able to inhibit biofilm formation and disassemble established S. aureus biofilms. We report here for the first time that both D- and L-isoforms of aspartate (Asp) inhibited S. aureus biofilm formation on tissue culture plates. Similar biofilm inhibition effects were also observed against other staphylococcal strains, including S. saprophyticus, S. equorum, S. chromogenes and S. haemolyticus. It was found that Asp at high concentrations (>10 mM) inhibited the growth of planktonic N315 cells, but at subinhibitory concentrations decreased the cellular metabolic activity without influencing cell growth. The decreased cellular metabolic activity might be the reason for the production of less protein and DNA in the matrix of the biofilms formed in the presence of Asp. However, varied inhibition efficacies of Asp were observed for biofilms formed by clinical staphylococcal isolates. There might be mechanisms other than decreasing the metabolic activity, e.g. the biofilm phenotypes, affecting biofilm formation in the presence of Asp. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  6. Protective effect of zinc aspartate against acetaminophen induced hepato-renal toxicity in albino rats

    International Nuclear Information System (INIS)

    Mohamed, E.T.; Said, A.I.; El-Sayed, S.A.

    2011-01-01

    Zinc is an essential nutrient that is required in humans and animals for many physiological functions, including antioxidant functions. The evidence to date indicates that zinc is an important element that links antioxidant system and tissue damage. Acetaminophen (AP), a widely used analgesic and antipyretic, produces hepatocyte and renal tubular necrosis in human and animals following overdose. In human, AP is one of the most common causes of acute liver failure as a result of accidental or deliberate overdose. Moreover, the initial event in AP toxicity is a toxic metabolic injury with the release of free radicals and subsequent cellular death by necrosis and apoptosis. This study was designed to evaluate the potential protective role of zinc aspartate in case of acetaminophen induced hepato-renal toxicity in rats. A total number of 32 adult male albino rats were divided into 4 equal groups: group I (control group), group II (zinc aspartate treated group), group III (acetaminophen treated group; by a single oral dose of 750 mg/kg body weight) and group IV acetaminophen plus zinc treated group; (zinc aspartate was intraperitoneally given one hour after acetaminophen administration in a dose of 30 mg/kg body weight). Serum levels of: alanine aminotransferase, aspartate aminotransferase, direct bilirubin, blood urea nitrogen, creatinine, uric acid, xanthine oxidase (XO), glutathione (GSH), malonaldehyde (MDA) and nitric oxide (NO) were assessed in all groups. The results of this study showed that treatment with acetaminophen alone (group III) produced a significant increase in serum levels of the liver enzymes and direct bilirubin. Moreover, in the same group there was a significant increase in the blood urea nitrogen and serum creatinine compared to the control group. In addition, there was a significant increase in XO and MDA and a significant decrease in GSH and NO level. Injection of rats with zinc aspartate after acetaminophen treatment could produce a

  7. Oligodendrocytes Do Not Export NAA-Derived Aspartate In Vitro.

    Science.gov (United States)

    I Amaral, Ana; Hadera, Mussie Ghezu; Kotter, Mark; Sonnewald, Ursula

    2017-03-01

    Oligodendroglial cells are known to de-acetylate the N-acetylaspartate (NAA) synthesized and released by neurons and use it for lipid synthesis. However, the role of NAA regarding their intermediary metabolism remains poorly understood. Two hypotheses were proposed regarding the fate of aspartate after being released by de-acetylation: (1) aspartate is metabolized in the mitochondria of oligodendrocyte lineage cells; (2) aspartate is released to the medium. We report here that aspartoacylase mRNA expression increases when primary rat oligodendrocyte progenitor cells (OPCs) differentiate into mature cells in culture. Moreover, characterising metabolic functions of acetyl coenzyme A and aspartate from NAA catabolism in mature oligodendrocyte cultures after 5 days using isotope-labelled glucose after 5-days of differentiation we found evidence of extensive NAA metabolism. Incubation with [1,6- 13 C]glucose followed by gas chromatography-mass spectrometry and high performance liquid chromatography analyses of cell extracts and media in the presence and absence of NAA established that the acetate moiety produced by hydrolysis of NAA does not enter mitochondrial metabolism in the form of acetyl coenzyme A. We also resolved the controversy concerning the possible release of aspartate to the medium: aspartate is not released to the medium by oligodendrocytes in amounts detectable by our methods. Therefore we propose that: aspartate released from NAA joins the cytosolic aspartate pool rapidly and takes part in the malate-aspartate shuttle, which transports reducing equivalents from glycolysis into the mitochondria for ATP production and enters the tricarboxylic acid cycle at a slow rate.

  8. Proteolytic crosstalk in multi-protease networks

    Science.gov (United States)

    Ogle, Curtis T.; Mather, William H.

    2016-04-01

    Processive proteases, such as ClpXP in E. coli, are conserved enzyme assemblies that can recognize and rapidly degrade proteins. These proteases are used for a number of purposes, including degrading mistranslated proteins and controlling cellular stress response. However, proteolytic machinery within the cell is limited in capacity and can lead to a bottleneck in protein degradation, whereby many proteins compete (‘queue’) for proteolytic resources. Previous work has demonstrated that such queueing can lead to pronounced statistical relationships between different protein counts when proteins compete for a single common protease. However, real cells contain many different proteases, e.g. ClpXP, ClpAP, and Lon in E. coli, and it is not clear how competition between proteins for multiple classes of protease would influence the dynamics of cellular networks. In the present work, we theoretically demonstrate that a multi-protease proteolytic bottleneck can substantially couple the dynamics for both simple and complex (oscillatory) networks, even between substrates with substantially different affinities for protease. For these networks, queueing often leads to strong positive correlations between protein counts, and these correlations are strongest near the queueing theoretic point of balance. Furthermore, we find that the qualitative behavior of these networks depends on the relative size of the absolute affinity of substrate to protease compared to the cross affinity of substrate to protease, leading in certain regimes to priority queue statistics.

  9. Cephalopod vision involves dicarboxylic amino acids: D-aspartate, L-aspartate and L-glutamate.

    Science.gov (United States)

    D'Aniello, Salvatore; Spinelli, Patrizia; Ferrandino, Gabriele; Peterson, Kevin; Tsesarskia, Mara; Fisher, George; D'Aniello, Antimo

    2005-03-01

    In the present study, we report the finding of high concentrations of D-Asp (D-aspartate) in the retina of the cephalopods Sepia officinalis, Loligo vulgaris and Octopus vulgaris. D-Asp increases in concentration in the retina and optic lobes as the animal develops. In neonatal S. officinalis, the concentration of D-Asp in the retina is 1.8+/-0.2 micromol/g of tissue, and in the optic lobes it is 5.5+/-0.4 micromol/g of tissue. In adult animals, D-Asp is found at a concentration of 3.5+/-0.4 micromol/g in retina and 16.2+/-1.5 micromol/g in optic lobes (1.9-fold increased in the retina, and 2.9-fold increased in the optic lobes). In the retina and optic lobes of S. officinalis, the concentration of D-Asp, L-Asp (L-aspartate) and L-Glu (L-glutamate) is significantly influenced by the light/dark environment. In adult animals left in the dark, these three amino acids fall significantly in concentration in both retina (approx. 25% less) and optic lobes (approx. 20% less) compared with the control animals (animals left in a diurnal/nocturnal physiological cycle). The reduction in concentration is in all cases statistically significant (P=0.01-0.05). Experiments conducted in S. officinalis by using D-[2,3-3H]Asp have shown that D-Asp is synthesized in the optic lobes and is then transported actively into the retina. D-aspartate racemase, an enzyme which converts L-Asp into D-Asp, is also present in these tissues, and it is significantly decreased in concentration in animals left for 5 days in the dark compared with control animals. Our hypothesis is that the dicarboxylic amino acids, D-Asp, L-Asp and L-Glu, play important roles in vision.

  10. Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy

    Science.gov (United States)

    2017-10-01

    AWARD NUMBER: W81XWH-16-1-0615 TITLE: Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy PRINCIPAL...29 Sep 2017 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Targeting Neutrophil Protease-Mediated Degradation of Tsp-1 to Induce Metastatic Dormancy...infection or cigarette smoke enhanced pulmonary metastasis from breast cancer in humans and mice. Similarly, autoimmune arthritis, characterized by

  11. Effect of the replacement of aspartic acid/glutamic acid residues with asparagine/glutamine residues in RNase He1 from Hericium erinaceus on inhibition of human leukemia cell line proliferation.

    Science.gov (United States)

    Kobayashi, Hiroko; Motoyoshi, Naomi; Itagaki, Tadashi; Suzuki, Mamoru; Inokuchi, Norio

    2015-01-01

    RNase He1 from Hericium erinaceus, a member of the RNase T1 family, has high identity with RNase Po1 from Pleurotus ostreatus with complete conservation of the catalytic sequence. However, the optimal pH for RNase He1 activity is lower than that of RNase Po1, and the enzyme shows little inhibition of human tumor cell proliferation. Hence, to investigate the potential antitumor activity of recombinant RNase He1 and to possibly enhance its optimum pH, we generated RNase He1 mutants by replacing 12 Asn/Gln residues with Asp/Glu residues; the amino acid sequence of RNase Po1 was taken as reference. These mutants were then expressed in Escherichia coli. Using site-directed mutagenesis, we successfully modified the optimal pH for enzyme activity and generated a recombinant RNase He1 that inhibited the proliferation of cells in the human leukemia cell line. These properties are extremely important in the production of anticancer biologics that are based on RNase activity.

  12. D-aspartate and NMDA, but not L-aspartate, block AMPA receptors in rat hippocampal neurons

    DEFF Research Database (Denmark)

    Gong, Xiang-Qun; Frandsen, Anne; Lu, Wei-Yang

    2005-01-01

    1 The amino acid, D-aspartate, exists in the mammalian brain and is an agonist at the N-methyl-D-aspartate (NMDA) subtype of ionotropic glutamate receptors. Here, for the first time, we studied the actions of D-aspartate on alpha-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate receptors (AMPARs......) in acutely isolated rat hippocampal neurons. 2 In the presence of the NMDA receptor channel blocker, MK801, D-aspartate inhibited kainate-induced AMPAR current in hippocampal neurons. The inhibitory action of D-aspartate on kainate-induced AMPAR current was concentration-dependent and was voltage......-independent in the tested voltage range (-80 to +60 mV). 3 The estimated EC50 of the L-glutamate-induced AMPAR current was increased in the presence of D-aspartate, while the estimated maximum L-glutamate-induced AMPAR current was not changed. D-aspartate concentration-dependently shifted the dose-response curve of kainate...

  13. Modulation of the epithelial sodium channel (ENaC by bacterial metalloproteases and protease inhibitors.

    Directory of Open Access Journals (Sweden)

    Michael B Butterworth

    Full Text Available The serralysin family of metalloproteases is associated with the virulence of multiple gram-negative human pathogens, including Pseudomonas aeruginosa and Serratia marcescens. The serralysin proteases share highly conserved catalytic domains and show evolutionary similarity to the mammalian matrix metalloproteases. Our previous studies demonstrated that alkaline protease (AP from Pseudomonas aeruginosa is capable of activating the epithelial sodium channel (ENaC, leading to an increase in sodium absorption in airway epithelia. The serralysin proteases are often co-expressed with endogenous, intracellular or periplasmic inhibitors, which putatively protect the bacterium from unwanted or unregulated protease activities. To evaluate the potential use of these small protein inhibitors in regulating the serralysin induced activation of ENaC, proteases from Pseudomonas aeruginosa and Serratia marcescens were purified for characterization along with a high affinity inhibitor from Pseudomonas. Both proteases showed activity against in vitro substrates and could be blocked by near stoichiometric concentrations of the inhibitor. In addition, both proteases were capable of activating ENaC when added to the apical surfaces of multiple epithelial cells with similar slow activation kinetics. The high-affinity periplasmic inhibitor from Pseudomonas effectively blocked this activation. These data suggest that multiple metalloproteases are capable of activating ENaC. Further, the endogenous, periplasmic bacterial inhibitors may be useful for modulating the downstream effects of the serralysin virulence factors under physiological conditions.

  14. Cross-talk between malarial cysteine proteases and falstatin: the BC loop as a hot-spot target.

    Directory of Open Access Journals (Sweden)

    Srinivasan Sundararaj

    Full Text Available Cysteine proteases play a crucial role in the development of the human malaria parasites Plasmodium falciparum and Plasmodium vivax. Our earlier studies demonstrated that these enzymes are equipped with specific domains for defined functions and further suggested the mechanism of activation of cysteine proteases. The activities of these proteases are regulated by a new class of endogenous inhibitors of cysteine proteases (ICPs. Structural studies of the ICPs of Trypanosoma cruzi (chagasin and Plasmodium berghei (PbICP indicated that three loops (termed BC, DE, and FG are crucial for binding to target proteases. Falstatin, an ICP of P. falciparum, appears to play a crucial role in invasion of erythrocytes and hepatocytes. However, the mechanism of inhibition of cysteine proteases by falstatin has not been established. Our study suggests that falstatin is the first known ICP to function as a multimeric protein. Using site-directed mutagenesis, hemoglobin hydrolysis assays and peptide inhibition studies, we demonstrate that the BC loop, but not the DE or FG loops, inhibits cysteine proteases of P. falciparum and P. vivax via hydrogen bonds. These results suggest that the BC loop of falstatin acts as a hot-spot target for inhibiting malarial cysteine proteases. This finding suggests new strategies for the development of anti-malarial agents based on protease-inhibitor interactions.

  15. NCBI nr-aa BLAST: CBRC-PMAR-01-0741 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-PMAR-01-0741 sp|Q53RT3|APRV1_HUMAN Retroviral-like aspartic protease 1 precursor (Skin...-specific retroviral-like aspartic protease) (Skin aspartic protease) (SASPase) (TPA-inducible aspar

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

    Directory of Open Access Journals (Sweden)

    Zongyun Chen

    development of diagnostic and therapeutic agents for human diseases that target diverse proteases.

  17. High-resolution structure of a retroviral protease folded as a monomer

    International Nuclear Information System (INIS)

    Gilski, Miroslaw; Kazmierczyk, Maciej; Krzywda, Szymon; Zábranská, Helena; Cooper, Seth; Popović, Zoran; Khatib, Firas; DiMaio, Frank; Thompson, James; Baker, David; Pichová, Iva; Jaskolski, Mariusz

    2011-01-01

    The crystal structure of Mason–Pfizer monkey virus protease folded as a monomer has been solved by molecular replacement using a model generated by players of the online game Foldit. The structure shows at high resolution the details of a retroviral protease folded as a monomer which can guide rational design of protease dimerization inhibitors as retroviral drugs. Mason–Pfizer monkey virus (M-PMV), a D-type retrovirus assembling in the cytoplasm, causes simian acquired immunodeficiency syndrome (SAIDS) in rhesus monkeys. Its pepsin-like aspartic protease (retropepsin) is an integral part of the expressed retroviral polyproteins. As in all retroviral life cycles, release and dimerization of the protease (PR) is strictly required for polyprotein processing and virion maturation. Biophysical and NMR studies have indicated that in the absence of substrates or inhibitors M-PMV PR should fold into a stable monomer, but the crystal structure of this protein could not be solved by molecular replacement despite countless attempts. Ultimately, a solution was obtained in mr-rosetta using a model constructed by players of the online protein-folding game Foldit. The structure indeed shows a monomeric protein, with the N- and C-termini completely disordered. On the other hand, the flap loop, which normally gates access to the active site of homodimeric retropepsins, is clearly traceable in the electron density. The flap has an unusual curled shape and a different orientation from both the open and closed states known from dimeric retropepsins. The overall fold of the protein follows the retropepsin canon, but the C α deviations are large and the active-site ‘DTG’ loop (here NTG) deviates up to 2.7 Å from the standard conformation. This structure of a monomeric retropepsin determined at high resolution (1.6 Å) provides important extra information for the design of dimerization inhibitors that might be developed as drugs for the treatment of retroviral infections

  18. Targeting cysteine proteases in trypanosomatid disease drug discovery.

    Science.gov (United States)

    Ferreira, Leonardo G; Andricopulo, Adriano D

    2017-12-01

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

  19. Non-enzymic beta-decarboxylation of aspartic acid.

    Science.gov (United States)

    Doctor, V. M.; Oro, J.

    1972-01-01

    Study of the mechanism of nonenzymic beta-decarboxylation of aspartic acid in the presence of metal ions and pyridoxal. The results suggest that aspartic acid is first converted to oxalacetic acid by transamination with pyridoxal which in turn is converted to pyridoxamine. This is followed by decarboxylation of oxalacetic acid to form pyruvic acid which transaminates with pyridoxamine to form alanine. The possible significance of these results to prebiotic molecular evolution is briefly discussed.

  20. Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum.

    Science.gov (United States)

    Castagliuolo, I; LaMont, J T; Nikulasson, S T; Pothoulakis, C

    1996-01-01

    Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis. We have previously reported that S. boulardii inhibits rat ileal secretion in response to C. difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C. Pothoulakis, C. P. Kelly, M. A. Joshi, N. Gao, C. J. O'Keane, I. Castagliuolo, and J. T. LaMont, Gastroenterology 104: 1108-1115, 1993). The aim of this study was to purify and characterize this protease. S. boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose. The effect of S. boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S. boulardii protease revealed a major band at 54 kDa. Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%). Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%). Preincubation of toxin A with S. boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A. Thus, S. boulardii protease inhibits the intestinal effects of C. difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor. Our results may be relevant to the mechanism by which S. boulardii exerts its protective effects in C. difficile infection in humans. PMID:8945570

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

    African Journals Online (AJOL)

    PRECIOUS

    2009-12-15

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

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

    African Journals Online (AJOL)

    user

    2013-03-20

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

  3. Current and Novel Inhibitors of HIV Protease

    Czech Academy of Sciences Publication Activity Database

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

    2009-01-01

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

  4. The action of neutrophil serine proteases on elastin and its precursor

    DEFF Research Database (Denmark)

    Heinz, Andrea; Jung, Michael C; Jahreis, Günther

    2012-01-01

    This study aimed to investigate the degradation of the natural substrates tropoelastin and elastin by the neutrophil-derived serine proteases human leukocyte elastase (HLE), proteinase 3 (PR3) and cathepsin G (CG). Focus was placed on determining their cleavage site specificities using mass...... spectrometric techniques. Moreover, the release of bioactive peptides from elastin by the three proteases was studied. Tropoelastin was comprehensively degraded by all three proteases, whereas less cleavage occurred in mature cross-linked elastin. An analysis of the cleavage site specificities of the three...... proteases in tropoelastin and elastin revealed that HLE and PR3 similarly tolerate hydrophobic and/or aliphatic amino acids such as Ala, Gly and Val at P(1), which are also preferred by CG. In addition, CG prefers the bulky hydrophobic amino acid Leu and accepts the bulky aromatic amino acids Phe and Tyr...

  5. Analysis of the interaction between the aspartic peptidase inhibitor SQAPI and aspartic peptidases using surface plasmon resonance.

    Science.gov (United States)

    Farley, Peter C; Christeller, John T; Sullivan, Michelle E; Sullivan, Patrick A; Laing, William A

    2002-01-01

    Aspartic peptidase inhibitors, which are themselves proteins, are strong inhibitors (small inhibition constants) of some aspartic peptidases but not others. However, there have been no studies of the kinetics of the interaction between a proteinaceous aspartic peptidase inhibitor and aspartic peptidases. This paper describes an analysis of rate constants for the interaction between recombinant squash aspartic peptidase inhibitor (rSQAPI) and a panel of aspartic peptidases that have a range of inhibition constants for SQAPI. Purified rSQAPI completely inhibits pepsin at a 1:1 molar ratio of pepsin to rSQAPI monomer (inhibition constant 1 nM). The interaction of pepsin with immobilized rSQAPI, at pH values between 3.0 and 6.0, was monitored using surface plasmon resonance. Binding of pepsin to rSQAPI was slow (association rate constants ca 10(4)M (-1)s(-1)), but rSQAPI was an effective pepsin inhibitor because dissociation of the rSQAPI-pepsin complex was much slower (dissociation rate constants ca 10(-4)s(-1)), especially at low pH values. Similar results were obtained with a His-tagged rSQAPI. Strong inhibition (inhibition constant 3 nM) of one isoform (rSap4) of the family of Candida albicans-secreted aspartic peptidases was, as with pepsin, characterized by slow binding of rSap4 and slower dissociation of the rSap4-inhibitor complex. In contrast, weaker inhibition of the Glomerella cingulata-secreted aspartic peptidase (inhibition constant 7 nM) and the C. albicans rSap1 and Sap2 isoenzymes (inhibition constants 25 and 400 nM, respectively) was, in each case, characterized by a larger dissociation rate constant. Copyright 2002 John Wiley & Sons, Ltd.

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

    Directory of Open Access Journals (Sweden)

    Byoung-Kuk Na

    2010-10-01

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

  7. Subject-driven titration of biphasic insulin aspart 30 twice daily is non-inferior to investigator-driven titration in Chinese patients with type 2 diabetes inadequately controlled with premixed human insulin: A randomized, open-label, parallel-group, multicenter trial.

    Science.gov (United States)

    Yang, Wenying; Zhu, Lvyun; Meng, Bangzhu; Liu, Yu; Wang, Wenhui; Ye, Shandong; Sun, Li; Miao, Heng; Guo, Lian; Wang, Zhanjian; Lv, Xiaofeng; Li, Quanmin; Ji, Qiuhe; Zhao, Weigang; Yang, Gangyi

    2016-01-01

    The present study was to compare the efficacy and safety of subject-driven and investigator-driven titration of biphasic insulin aspart 30 (BIAsp 30) twice daily (BID). In this 20-week, randomized, open-label, two-group parallel, multicenter trial, Chinese patients with type 2 diabetes inadequately controlled by premixed/self-mixed human insulin were randomized 1:1 to subject-driven or investigator-driven titration of BIAsp 30 BID, in combination with metformin and/or α-glucosidase inhibitors. Dose adjustment was decided by patients in the subject-driven group after training, and by investigators in the investigator-driven group. Eligible adults (n = 344) were randomized in the study. The estimated glycated hemoglobin (HbA1c) reduction was 14.5 mmol/mol (1.33%) in the subject-driven group and 14.3 mmol/mol (1.31%) in the investigator-driven group. Non-inferiority of subject-titration vs investigator-titration in reducing HbA1c was confirmed, with estimated treatment difference -0.26 mmol/mol (95% confidence interval -2.05, 1.53) (-0.02%, 95% confidence interval -0.19, 0.14). Fasting plasma glucose, postprandial glucose increment and self-measured plasma glucose were improved in both groups without statistically significant differences. One severe hypoglycemic event was experienced by one subject in each group. A similar rate of nocturnal hypoglycemia (events/patient-year) was reported in the subject-driven (1.10) and investigator-driven (1.32) groups. There were 64.5 and 58.1% patients achieving HbA1c titration of BIAsp 30 BID was as efficacious and well-tolerated as investigator-titration. The present study supported patients to self-titrate BIAsp 30 BID under physicians' supervision.

  8. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    Directory of Open Access Journals (Sweden)

    Werner Smidt

    Full Text Available The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1 infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS. Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  9. Potential elucidation of a novel CTL epitope in HIV-1 protease by the protease inhibitor resistance mutation L90M.

    Science.gov (United States)

    Smidt, Werner

    2013-01-01

    The combination of host immune responses and use of antiretrovirals facilitate partial control of human immunodeficiency virus type 1 (HIV-1) infection and result in delayed progression to Acquired Immunodeficiency Syndrome (AIDS). Both treatment and host immunity impose selection pressures on the highly mutable HIV-1 genome resulting in antiretroviral resistance and immune escape. Researchers have shown that antiretroviral resistance mutations can shape cytotoxic T-lymphocyte immunity by altering the epitope repertoire of HIV infected cells. Here it was discovered that an important antiretroviral resistance mutation, L90M in HIV protease, occurs at lower frequencies in hosts that harbor the B*15, B*48 or A*32 human leukocyte antigen subtypes. A likely reason is the elucidation of novel epitopes by L90M. NetMHCPan predictions reveal increased affinity of the peptide spanning the HIV protease region, PR 89-97 and PR 90-99 to HLA-B*15/B*48 and HLA-A*32 respectively due to the L90M substitution. The higher affinity could increase the chance of the epitope being presented and recognized by Cytotoxic T-lymphocytes and perhaps provide additional immunological pressures in the presence of antiretroviral attenuating mutations. This evidence supports the notion that knowledge of HLA allotypes in HIV infected individuals could augment antiretroviral treatment by the elucidation of epitopes due to antiretroviral resistance mutations in HIV protease.

  10. Genome-wide analysis of regulatory proteases sequences identified through bioinformatics data mining in Taenia solium.

    Science.gov (United States)

    Yan, Hong-Bin; Lou, Zhong-Zi; Li, Li; Brindley, Paul J; Zheng, Yadong; Luo, Xuenong; Hou, Junling; Guo, Aijiang; Jia, Wan-Zhong; Cai, Xuepeng

    2014-06-04

    Cysticercosis remains a major neglected tropical disease of humanity in many regions, especially in sub-Saharan Africa, Central America and elsewhere. Owing to the emerging drug resistance and the inability of current drugs to prevent re-infection, identification of novel vaccines and chemotherapeutic agents against Taenia solium and related helminth pathogens is a public health priority. The T. solium genome and the predicted proteome were reported recently, providing a wealth of information from which new interventional targets might be identified. In order to characterize and classify the entire repertoire of protease-encoding genes of T. solium, which act fundamental biological roles in all life processes, we analyzed the predicted proteins of this cestode through a combination of bioinformatics tools. Functional annotation was performed to yield insights into the signaling processes relevant to the complex developmental cycle of this tapeworm and to highlight a suite of the proteases as potential intervention targets. Within the genome of this helminth parasite, we identified 200 open reading frames encoding proteases from five clans, which correspond to 1.68% of the 11,902 protein-encoding genes predicted to be present in its genome. These proteases include calpains, cytosolic, mitochondrial signal peptidases, ubiquitylation related proteins, and others. Many not only show significant similarity to proteases in the Conserved Domain Database but have conserved active sites and catalytic domains. KEGG Automatic Annotation Server (KAAS) analysis indicated that ~60% of these proteases share strong sequence identities with proteins of the KEGG database, which are involved in human disease, metabolic pathways, genetic information processes, cellular processes, environmental information processes and organismal systems. Also, we identified signal peptides and transmembrane helices through comparative analysis with classes of important regulatory proteases

  11. A biotechnology perspective of fungal proteases

    Directory of Open Access Journals (Sweden)

    Paula Monteiro de Souza

    2015-06-01

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

  12. Natural inhibitors of tumor-associated proteases

    International Nuclear Information System (INIS)

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

    2002-01-01

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

  13. Extracellular proteases of Trichoderma species. A review.

    Science.gov (United States)

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

    2005-01-01

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

  14. Gut proteases target Yersinia invasin in vivo

    Directory of Open Access Journals (Sweden)

    Freund Sandra

    2011-04-01

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

  15. Combination of aspartic acid and glutamic acid inhibits tumor cell proliferation.

    Science.gov (United States)

    Yamaguchi, Yoshie; Yamamoto, Katsunori; Sato, Yoshinori; Inoue, Shinjiro; Morinaga, Tetsuo; Hirano, Eiichi

    2016-01-01

    Placental extract contains several biologically active compounds, and pharmacological induction of placental extract has therapeutic effects, such as improving liver function in patients with hepatitis or cirrhosis. Here, we searched for novel molecules with an anti-tumor activity in placental extracts. Active molecules were separated by chromatographic analysis, and their antiproliferative activities were determined by a colorimetric assay. We identified aspartic acid and glutamic acid to possess the antiproliferative activity against human hepatoma cells. Furthermore, we showed that the combination of aspartic acid and glutamic acid exhibited enhanced antiproliferative activity, and inhibited Akt phosphorylation. We also examined in vivo tumor inhibition activity using the rabbit VX2 liver tumor model. The treatment mixture (emulsion of the amino acids with Lipiodol) administered by hepatic artery injection inhibited tumor cell growth of the rabbit VX2 liver. These results suggest that the combination of aspartic acid and glutamic acid may be useful for induction of tumor cell death, and has the potential for clinical use as a cancer therapeutic agent.

  16. New insights into the metabolism of aspartate-family amino acids in plant seeds.

    Science.gov (United States)

    Wang, Wenyi; Xu, Mengyun; Wang, Guoping; Galili, Gad

    2018-02-05

    Aspartate-family amino acids. Aspartate (Asp)-family pathway, via several metabolic branches, leads to four key essential amino acids: Lys, Met, Thr, and Ile. Among these, Lys and Met have received the most attention, as they are the most limiting amino acid in cereals and legumes crops, respectively. The metabolic pathways of these four essential amino acids and their interactions with regulatory networks have been well characterized. Using this knowledge, extensive efforts have been devoted to augmenting the levels of these amino acids in various plant organs, especially seeds, which serve as the main source of human food and livestock feed. Seeds store a number of storage proteins, which are utilized as nutrient and energy resources. Storage proteins are composed of amino acids, to guarantee the continuation of plant progeny. Thus, understanding the seed metabolism, especially with respect to the accumulation of aspartate-derived amino acids Lys and Met, is a crucial factor for sustainable agriculture. In this review, we summarized the Asp-family pathway, with some new examples of accumulated Asp-family amino acids, particularly Lys and Met, in plant seeds. We also discuss the recent advances in understanding the roles of Asp-family amino acids during seed development.

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

    Directory of Open Access Journals (Sweden)

    Laura Pirisinu

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

  18. Age estimation in forensic sciences: application of combined aspartic acid racemization and radiocarbon analysis.

    Science.gov (United States)

    Alkass, Kanar; Buchholz, Bruce A; Ohtani, Susumu; Yamamoto, Toshiharu; Druid, Henrik; Spalding, Kirsty L

    2010-05-01

    Age determination of unknown human bodies is important in the setting of a crime investigation or a mass disaster because the age at death, birth date, and year of death as well as gender can guide investigators to the correct identity among a large number of possible matches. Traditional morphological methods used by anthropologists to determine age are often imprecise, whereas chemical analysis of tooth dentin, such as aspartic acid racemization, has shown reproducible and more precise results. In this study, we analyzed teeth from Swedish individuals using both aspartic acid racemization and radiocarbon methodologies. The rationale behind using radiocarbon analysis is that aboveground testing of nuclear weapons during the cold war (1955-1963) caused an extreme increase in global levels of carbon-14 ((14)C), which has been carefully recorded over time. Forty-four teeth from 41 individuals were analyzed using aspartic acid racemization analysis of tooth crown dentin or radiocarbon analysis of enamel, and 10 of these were split and subjected to both radiocarbon and racemization analysis. Combined analysis showed that the two methods correlated well (R(2) = 0.66, p Aspartic acid racemization also showed a good precision with an overall absolute error of 5.4 +/- 4.2 years. Whereas radiocarbon analysis gives an estimated year of birth, racemization analysis indicates the chronological age of the individual at the time of death. We show how these methods in combination can also assist in the estimation of date of death of an unidentified victim. This strategy can be of significant assistance in forensic casework involving dead victim identification.

  19. Age estimation in forensic sciences: Application of combined aspartic acid racemization and radiocarbon analysis

    Energy Technology Data Exchange (ETDEWEB)

    Alkass, K; Buchholz, B A; Ohtani, S; Yamamoto, T; Druid, H; Spalding, S L

    2009-11-02

    Age determination of unknown human bodies is important in the setting of a crime investigation or a mass disaster, since the age at death, birth date and year of death, as well as gender, can guide investigators to the correct identity among a large number of possible matches. Traditional morphological methods used by anthropologists to determine age are often imprecise, whereas chemical analysis of tooth dentin, such as aspartic acid racemization has shown reproducible and more precise results. In this paper we analyze teeth from Swedish individuals using both aspartic acid racemization and radiocarbon methodologies. The rationale behind using radiocarbon analysis is that above-ground testing of nuclear weapons during the cold war (1955-1963) caused an extreme increase in global levels of carbon-14 ({sup 14}C) which have been carefully recorded over time. Forty-four teeth from 41 individuals were analyzed using aspartic acid racemization analysis of tooth crown dentin or radiocarbon analysis of enamel and ten of these were split and subjected to both radiocarbon and racemization analysis. Combined analysis showed that the two methods correlated well (R2=0.66, p < 0.05). Radiocarbon analysis showed an excellent precision with an overall absolute error of 0.6 {+-} 04 years. Aspartic acid racemization also showed a good precision with an overall absolute error of 5.4 {+-} 4.2 years. Whereas radiocarbon analysis gives an estimated year of birth, racemization analysis indicates the chronological age of the individual at the time of death. We show how these methods in combination can also assist in the estimation of date of death of an unidentified victim. This strategy can be of significant assistance in forensic casework involving dead victim identification.

  20. Controversial Effects of D-Amino Acid Oxidase Activator (DAOA)/G72 on D-Amino Acid Oxidase (DAO) Activity in Human Neuronal, Astrocyte and Kidney Cell Lines: The N-methyl D-aspartate (NMDA) Receptor Hypofunction Point of View.

    Science.gov (United States)

    Jagannath, Vinita; Brotzakis, Zacharias Faidon; Parrinello, Michele; Walitza, Susanne; Grünblatt, Edna

    2017-01-01

    Dysfunction of D-amino acid oxidase ( DAO ) and DAO activator ( DAOA )/ G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA) receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y), astrocyte-like (1321N1) and kidney-like (HEK293) human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO) showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD)] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD), which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

  1. Controversial Effects of D-Amino Acid Oxidase Activator (DAOA/G72 on D-Amino Acid Oxidase (DAO Activity in Human Neuronal, Astrocyte and Kidney Cell Lines: The N-methyl D-aspartate (NMDA Receptor Hypofunction Point of View

    Directory of Open Access Journals (Sweden)

    Vinita Jagannath

    2017-10-01

    Full Text Available Dysfunction of D-amino acid oxidase (DAO and DAO activator (DAOA/G72 genes have been linked to neuropsychiatric disorders. The glutamate hypothesis of schizophrenia has proposed that increased DAO activity leads to decreased D-serine, which subsequently may lead to N-methyl-D-aspartate (NMDA receptor hypofunction. It has been shown that DAOA binds to DAO and increases its activity. However, there are also studies showing DAOA decreases DAO activity. Thus, the effect of DAOA on DAO is controversial. We aimed to understand the effect of DAOA on DAO activity in neuron-like (SH-SY5Y, astrocyte-like (1321N1 and kidney-like (HEK293 human cell lines. DAO activity was measured based on the release of hydrogen peroxide and its interaction with Amplex Red reagent. We found that DAOA increases DAO activity only in HEK293 cells, but has no effect on DAO activity in SH-SY5Y and 1321N1 cells. This might be because of different signaling pathways, or due to lower DAO and DAOA expression in SH-SY5Y and 1321N1 cells compared to HEK293 cells, but also due to different compartmentalization of the proteins. The lower DAO and DAOA expression in neuron-like SH-SY5Y and astrocyte-like 1321N1 cells might be due to tightly regulated expression, as previously reported in the human post-mortem brain. Our simulation experiments to demonstrate the interaction between DAOA and human DAO (hDAO showed that hDAO holoenzyme [hDAO with flavine adenine dinucleotide (FAD] becomes more flexible and misfolded in the presence of DAOA, whereas DAOA had no effect on hDAO apoprotein (hDAO without FAD, which indicate that DAOA inactivates hDAO holoenzyme. Furthermore, patch-clamp analysis demonstrated no effect of DAOA on NMDA receptor activity in NR1/NR2A HEK293 cells. In summary, the interaction between DAO and DAOA seems to be cell type and its biochemical characteristics dependent which still needs to be elucidated.

  2. Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins.

    Science.gov (United States)

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

    2011-02-01

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

  3. L-ornithine-L-aspartate infusion efficacy in hepatic encephalopathy

    International Nuclear Information System (INIS)

    Ahmad, I.

    2008-01-01

    To determine the efficacy of L-ornithine-L-aspartate in treatment of hepatic encephalopathy. Cirrhotic patients with hyperammonemia and overt hepatic encephalopathy were enrolled. Eighty patients were randomized to two treatment groups, L-ornithine-L-aspartate (20g/d) or placebo, both dissolved in 250mL of 5% dextrose water and infused intravenously for four hours a day for five consecutive days with 0.5 g/kg dietary protein intake at the end of daily treatment period. Outcome variables were postprandial blood ammonia and mental state grade. Adverse reactions and mortality were also determined. Both treatment groups were comparable regarding age, gender, etiology of cirrhosis, Child-Pugh class, mental state grade and blood ammonia at baseline. Although, improvement occurred in both groups, there was a greater improvement in L-ornithine-L-aspartate group with regard to both variables. Four patients in the placebo group and 2 in L-ornithine-L-aspartate group died. L-ornithine-L-aspartate infusions were found to be effective in cirrhotic patients with hepatic encephalopathy. (author)

  4. Structural transitions in crystals of native aspartate carbamoyltransferase

    International Nuclear Information System (INIS)

    Gouaux, J.E.; Lipscomb, W.N.

    1989-01-01

    Screened precession x-ray photographs of crystals of native aspartate carbamoyltransferase ligated with L-aspartate and phosphate reveal the presence of a crystal unit-cell dimension that is intermediate between the T (tense) and R (relaxed) states. Characterizing the intermediate (I) crystal is a c-axis unit-cell dimension of 149 angstrom, halfway between the c-axis length of the T (c = 142 angstrom) and R (c = 156 angstrom) states, in the space group P321. Preservation of the P321 space group indicates that the intermediate crystal form retains a threefold axis of symmetry, and therefore the enzyme has at minimum a threefold axis; however, it is not known whether the molecular twofold axis is conserved. The I crystals are formed by soaking T-state crystals with L-aspartate and phosphate. By raising the concentration of L-aspartate the authors can further transform the I crystals, without fragmentation, to a form that has the same unit-cell dimensions as R-state crystals grown in the presence of N-(phosphonoacetyl)-L-aspartate

  5. Purification and characterization of a milk-clotting aspartic proteinase from globe artichoke (Cynara scolymus L.).

    Science.gov (United States)

    Llorente, Berta E; Brutti, Cristina B; Caffini, Néstor O

    2004-12-29

    The study of proteinase expression in crude extracts from different organs of the globe artichoke (Cynara scolymus L.) disclosed that enzymes with proteolytic and milk-clotting activity are mainly located in mature flowers. Maximum proteolytic activity was recorded at pH 5.0, and inhibition studies showed that only pepstatin, specific for aspartic proteinases, presented a significant inhibitory effect. Such properties, in addition to easy enzyme inactivation by moderate heating, make this crude protease extract potentially useful for cheese production. Adsorption with activated carbon, together with anion exchange and affinity chromatography, led to the isolation of a heterodimeric milk-clotting proteinase consisting of 30- and 15-kDa subunits. MALDI-TOF MS of the 15-kDa chain determined a 15.358-Da mass, and the terminal amino sequence presented 96% homology with the smaller cardosin A subunit. The amino terminal sequence of the 30-kDa chain proved to be identical to the larger cardosin A subunit. Electrophoresis evidenced proteinase self-processing that was confirmed by immunoblots presenting 62-, 30-, and 15-kDa bands.

  6. Correlation between myocardial malate/aspartate shuttle activity and EAAT1 protein expression in hyper- and hypothyroidism.

    Science.gov (United States)

    Ralphe, J Carter; Bedell, Kurt; Segar, Jeffrey L; Scholz, Thomas D

    2005-05-01

    In the heart, elevated thyroid hormone leads to upregulation of metabolic pathways associated with energy production and development of hypertrophy. The malate/aspartate shuttle, which transfers cytosolic-reducing equivalents into the cardiac mitochondria, is increased 33% in hyperthyroid rats. Within the shuttle, the aspartate-glutamate carrier is rate limiting. The excitatory amino acid transporter type 1 (EAAT1) functions as a glutamate carrier in the malate/aspartate shuttle. In this study, we hypothesize that EAAT1 is regulated by thyroid hormone. Adult rats were injected with triiodothyronine (T3) or saline over a period of 8-9 days or provided with propylthiouracil (PTU) in their drinking water for 2 mo. Steady-state mRNA levels of EAAT1 and aralar1 and citrin (both cardiac mitochondrial aspartate-glutamate transporters) were determined by Northern blot analysis and normalized to 18S rRNA. A spectrophotometric assay of maximal malate/aspartate shuttle activity was performed on isolated cardiac mitochondria from PTU-treated and control animals. Protein lysates from mitochondria were separated by SDS-PAGE and probed with a human anti-EAAT1 IgG. Compared with control, EAAT1 mRNA levels (arbitrary units) were increased nearly threefold in T3-treated (3.1 +/- 0.5 vs. 1.1 +/- 0.2; P Hyperthyroidism in rats is related to an increase in cardiac expression of EAAT1 mRNA and protein. The 49% increase in EAAT1 mitochondrial protein level shows that malate/aspartate shuttle activity increased in hyperthyroid rat cardiac mitochondria. Although hypothyroidism resulted in a decrease in EAAT1 mRNA, neither the EAAT1 protein level nor shuttle activity was affected. EAAT1 regulation by thyroid hormone may facilitate increased metabolic demands of the cardiomyocyte during hyperthyroidism and impact cardiac function in hyperthyroidism.

  7. A New Pepstatin-Insensitive Thermopsin-Like Protease Overproduced in Peptide-Rich Cultures of Sulfolobus solfataricus

    Directory of Open Access Journals (Sweden)

    Marta Gogliettino

    2014-02-01

    Full Text Available In this study, we gain insight into the extracellular proteolytic system of Sulfolobus solfataricus grown on proteinaceous substrates, providing further evidence that acidic proteases were specifically produced in response to peptide-rich media. The main proteolytic component was the previously isolated SsMTP (Sulfolobus solfataricus multi-domain thermopsin-like protease, while the less abundant (named SsMTP-1 one was purified, characterized and identified as the sso1175 gene-product. The protein revealed a multi-domain organization shared with the cognate SsMTP with a catalytic domain followed by several tandemly-repeated motifs. Moreover, both enzymes were found spread across the Crenarchaeota phylum and belonging to the thermopsin family, although segregated into diverse phylogenetic clusters. SsMTP-1 showed a 75-kDa molecular mass and was stable in the temperature range 50–90 °C, with optimal activity at 70 °C and pH 2.0. Serine, metallo and aspartic protease inhibitors did not affect the enzyme activity, designating SsMTP-1 as a new member of the pepstatin-insensitive aspartic protease family. The peptide-bond-specificity of SsMTP-1 in the cleavage of the oxidized insulin B chain was uncommon amongst thermopsins, suggesting that it could play a distinct, but cooperative role in the protein degradation machinery. Interestingly, predictions of the transmembrane protein topology of SsMTP and SsMTP-1 strongly suggest a possible contribution in signal-transduction pathways.

  8. Biodegradability and tissue reaction of random copolymers of L-leucine, L-aspartic acid, and L-aspartic acid esters

    NARCIS (Netherlands)

    Marck, K.W.; Wildevuur, Ch.R.H.; Sederel, W.L.; Bantjes, A.; Feijen, Jan

    1977-01-01

    A series of copoly(α-amino acids) with varying percentages of hydrophilic (l-aspartic acid) and hydrophobic monomers (l-leucine, ß-methyl-l-aspartate, and ß-benzyl-l-aspartate) were implanted subcutaneously in rats and the macroscopic degradation behavior was studied. Three groups of materials (A,

  9. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

    Directory of Open Access Journals (Sweden)

    Rok Gaber

    2013-11-01

    Full Text Available To effectively fight against the human immunodeficiency virus infection/ acquired immunodeficiency syndrome (HIV/AIDS epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity.

  10. Noninvasive High-Throughput Single-Cell Analysis of HIV Protease Activity Using Ratiometric Flow Cytometry

    Science.gov (United States)

    Gaber, Rok; Majerle, Andreja; Jerala, Roman; Benčina, Mojca

    2013-01-01

    To effectively fight against the human immunodeficiency virus infection/acquired immunodeficiency syndrome (HIV/AIDS) epidemic, ongoing development of novel HIV protease inhibitors is required. Inexpensive high-throughput screening assays are needed to quickly scan large sets of chemicals for potential inhibitors. We have developed a Förster resonance energy transfer (FRET)-based, HIV protease-sensitive sensor using a combination of a fluorescent protein pair, namely mCerulean and mCitrine. Through extensive in vitro characterization, we show that the FRET-HIV sensor can be used in HIV protease screening assays. Furthermore, we have used the FRET-HIV sensor for intracellular quantitative detection of HIV protease activity in living cells, which more closely resembles an actual viral infection than an in vitro assay. We have developed a high-throughput method that employs a ratiometric flow cytometry for analyzing large populations of cells that express the FRET-HIV sensor. The method enables FRET measurement of single cells with high sensitivity and speed and should be used when subpopulation-specific intracellular activity of HIV protease needs to be estimated. In addition, we have used a confocal microscopy sensitized emission FRET technique to evaluate the usefulness of the FRET-HIV sensor for spatiotemporal detection of intracellular HIV protease activity. PMID:24287545

  11. The Mitochondrial m-AAA Protease Prevents Demyelination and Hair Greying.

    Science.gov (United States)

    Wang, Shuaiyu; Jacquemyn, Julie; Murru, Sara; Martinelli, Paola; Barth, Esther; Langer, Thomas; Niessen, Carien M; Rugarli, Elena I

    2016-12-01

    The m-AAA protease preserves proteostasis of the inner mitochondrial membrane. It ensures a functional respiratory chain, by controlling the turnover of respiratory complex subunits and allowing mitochondrial translation, but other functions in mitochondria are conceivable. Mutations in genes encoding subunits of the m-AAA protease have been linked to various neurodegenerative diseases in humans, such as hereditary spastic paraplegia and spinocerebellar ataxia. While essential functions of the m-AAA protease for neuronal survival have been established, its role in adult glial cells remains enigmatic. Here, we show that deletion of the highly expressed subunit AFG3L2 in mature mouse oligodendrocytes provokes early-on mitochondrial fragmentation and swelling, as previously shown in neurons, but causes only late-onset motor defects and myelin abnormalities. In contrast, total ablation of the m-AAA protease, by deleting both Afg3l2 and its paralogue Afg3l1, triggers progressive motor dysfunction and demyelination, owing to rapid oligodendrocyte cell death. Surprisingly, the mice showed premature hair greying, caused by progressive loss of melanoblasts that share a common developmental origin with Schwann cells and are targeted in our experiments. Thus, while both neurons and glial cells are dependant on the m-AAA protease for survival in vivo, complete ablation of the complex is necessary to trigger death of oligodendrocytes, hinting to cell-autonomous thresholds of vulnerability to m-AAA protease deficiency.

  12. Resonant electron capture by aspartame and aspartic acid molecules.

    Science.gov (United States)

    Muftakhov, M V; Shchukin, P V

    2016-12-30

    The processes for dissociative electron capture are the key mechanisms for decomposition of biomolecules, proteins in particular, under interaction with low-energy electrons. Molecules of aspartic acid and aspartame, i.e. modified dipeptides, were studied herein to define the impact of the side functional groups on peptide chain decomposition in resonant electron-molecular reactions. The processes of formation and decomposition of negative ions of both aspartame and aspartic acid were studied by mass spectrometry of negative ions under resonant electron capture. The obtained mass spectra were interpreted under thermochemical analysis by quantum chemical calculations. Main channels of negative molecular ions fragmentation were found and characteristic fragment ions were identified. The СООН fragment of the side chain in aspartic acid is shown to play a key role like the carboxyl group in amino acids and aliphatic oligopeptides. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  13. Moringa oleifera Lam.: Protease activity against blood coagulation cascade.

    Science.gov (United States)

    Satish, A; Sairam, Sudha; Ahmed, Faiyaz; Urooj, Asna

    2012-01-01

    The present study evaluated the protease activity of aqueous extracts of Moringa oleifera (Moringaceae) leaf (MOL) and root (MOR). Protease activity was assayed using casein, human plasma clot and human fibrinogen as substrates. Caseinolytic activity of MOL was significantly higher (P ≤ 0.05) than that of MOR. Similar observations were found in case of human plasma clot hydrolyzing activity, wherein MOL caused significantly higher (P ≤ 0.05) plasma clot hydrolysis than MOR. Zymographic techniques were used to detect proteolytic enzymes following electrophoretic separation in gels. Further, both the extracts exhibited significant procoagulant activity as reflected by a significant decrease (P ≤ 0.05) in recalcification time, accompanied by fibrinogenolytic and fibrinolytic activities; clotting time was decreased from 180 ± 10 sec to 119 ± 8 sec and 143 ± 10 sec by MOL and MOR, respectively, at a concentration of 2.5 mg/mL. Fibrinogenolytic (human fibrinogen) and fibrinolytic activity (human plasma clot) was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), plate method and colorimetric method. Zymographic profile indicated that both the extracts exerted their procoagulant activity by selectively hydrolyzing Aα and Bβ subunits of fibrinogen to form fibrin clot, thereby exhibiting fibrinogenolytic activity. However, prolonged incubation resulted in degradation of the formed fibrin clot, suggesting fibrinolytic like activity. These findings support the traditional usage of M. oleifera extracts for wound healing.

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

    Directory of Open Access Journals (Sweden)

    Landys A. Lopez Quezada

    2011-06-01

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

  15. Proteolytic activity in the adult and larval stages of the human roundworm parasite Angiostrongylus costaricensis

    Directory of Open Access Journals (Sweden)

    Karina Mastropasqua Rebello

    2012-09-01

    Full Text Available Angiostrongylus costaricensis is a nematode that causes abdominal angiostrongyliasis, a widespread human parasitism in Latin America. This study aimed to characterize the protease profiles of different developmental stages of this helminth. First-stage larvae (L1 were obtained from the faeces of infected Sigmodon hispidus rodents and third-stage larvae (L3 were collected from mollusks Biomphalaria glabrata previously infected with L1. Adult worms were recovered from rodent mesenteric arteries. Protein extraction was performed after repeated freeze-thaw cycles followed by maceration of the nematodes in 40 mM Tris base. Proteolysis of gelatin was observed by zymography and found only in the larval stages. In L3, the gelatinolytic activity was effectively inhibited by orthophenanthroline, indicating the involvement of metalloproteases. The mechanistic class of the gelatinases from L1 could not be precisely determined using traditional class-specific inhibitors. Adult worm extracts were able to hydrolyze haemoglobin in solution, although no activity was observed by zymography. This haemoglobinolytic activity was ascribed to aspartic proteases following its effective inhibition by pepstatin, which also inhibited the haemoglobinolytic activity of L1 and L3 extracts. The characterization of protease expression throughout the A. costaricensis life cycle may reveal key factors influencing the process of parasitic infection and thus foster our understanding of the disease pathogenesis.

  16. Activation of ADAM 12 protease by copper

    DEFF Research Database (Denmark)

    Loechel, F; Wewer, Ulla M.

    2001-01-01

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

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

    African Journals Online (AJOL)

    SERVER

    2008-04-17

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

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

    African Journals Online (AJOL)

    USER

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

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

    African Journals Online (AJOL)

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

  20. Coordination features and use of aspartic acid in chelatometry

    International Nuclear Information System (INIS)

    Sergeev, G.M.; Korenman, I.M.

    1978-01-01

    Considered are coordination peculiarities and application of aspartic and as selective reagent for Be(2) and Mo(6) in chelatometry. pH range of the complexes with aspartic acid for Be(2), pH 4-9, for Mo(6), pH 3-9 are determined. Stability constants of the complexes are found. These values can serve as the basis for selective determination of Be(2) and Mo(6) with asparic acid, which are not always successful with EDTA and DTPA

  1. Identification of novel malarial cysteine protease inhibitors using structure-based virtual screening of a focused cysteine protease inhibitor library.

    Science.gov (United States)

    Shah, Falgun; Mukherjee, Prasenjit; Gut, Jiri; Legac, Jennifer; Rosenthal, Philip J; Tekwani, Babu L; Avery, Mitchell A

    2011-04-25

    Malaria, in particular that caused by Plasmodium falciparum , is prevalent across the tropics, and its medicinal control is limited by widespread drug resistance. Cysteine proteases of P. falciparum , falcipain-2 (FP-2) and falcipain-3 (FP-3), are major hemoglobinases, validated as potential antimalarial drug targets. Structure-based virtual screening of a focused cysteine protease inhibitor library built with soft rather than hard electrophiles was performed against an X-ray crystal structure of FP-2 using the Glide docking program. An enrichment study was performed to select a suitable scoring function and to retrieve potential candidates against FP-2 from a large chemical database. Biological evaluation of 50 selected compounds identified 21 diverse nonpeptidic inhibitors of FP-2 with a hit rate of 42%. Atomic Fukui indices were used to predict the most electrophilic center and its electrophilicity in the identified hits. Comparison of predicted electrophilicity of electrophiles in identified hits with those in known irreversible inhibitors suggested the soft-nature of electrophiles in the selected target compounds. The present study highlights the importance of focused libraries and enrichment studies in structure-based virtual screening. In addition, few compounds were screened against homologous human cysteine proteases for selectivity analysis. Further evaluation of structure-activity relationships around these nonpeptidic scaffolds could help in the development of selective leads for antimalarial chemotherapy.

  2. Crystal structure of caesium hydrogen (L)-aspartate and an overview of crystalline compounds of aspartic acid with inorganic constituents

    Energy Technology Data Exchange (ETDEWEB)

    Fleck, M. [Universitaet Wien (Austria). Institut fuer Mineralogie und Kristallographie; Emmerich, R.; Bohaty, L. [Universitaet zu Koeln (Austria). Institut fuer Kristallographie

    2010-08-15

    The crystal structure of the new polar compound caesium hydrogen (L)-aspartate, Cs(C{sub 4}H{sub 6}NO{sub 4}), (abbreviated: Cs(L -AspH)) was determined from single crystal X-ray diffraction data; it comprises two crystallographically different L -AspH anions that are connected via caesium cations to form a three dimensional framework. The Cs ions are irregularly sevenfold[Cs1O{sub 7}] respectively eightfold[Cs2O{sub 8}] coordinated to all {alpha}- and {beta}- carboxylate oxygen atoms. Cs(L -AspH) represents a novel structure type of its own, as do most compounds of (L)-aspartic acid with inorganic constituents. A brief summary of such structurally known aspartates is given. (copyright 2010 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim) (orig.)

  3. TMPRSS12 Is an Activating Protease for Subtype B Avian Metapneumovirus.

    Science.gov (United States)

    Yun, Bingling; Zhang, Yao; Liu, Yongzhen; Guan, Xiaolu; Wang, Yongqiang; Qi, Xiaole; Cui, Hongyu; Liu, Changjun; Zhang, Yanping; Gao, Honglei; Gao, Li; Li, Kai; Gao, Yulong; Wang, Xiaomei

    2016-12-15

    The entry of avian metapneumovirus (aMPV) into host cells initially requires the fusion of viral and cell membranes, which is exclusively mediated by fusion (F) protein. Proteolysis of aMPV F protein by endogenous proteases of host cells allows F protein to induce membrane fusion; however, these proteases have not been identified. Here, we provide the first evidence that the transmembrane serine protease TMPRSS12 facilitates the cleavage of subtype B aMPV (aMPV/B) F protein. We found that overexpression of TMPRSS12 enhanced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. Subsequently, knockdown of TMPRSS12 with specific small interfering RNAs (siRNAs) reduced aMPV/B F protein cleavage, F protein fusogenicity, and viral replication. We also found a cleavage motif in the aMPV/B F protein (amino acids 100 and 101) that was recognized by TMPRSS12. The histidine, aspartic acid, and serine residue (HDS) triad of TMPRSS12 was shown to be essential for the proteolysis of aMPV/B F protein via mutation analysis. Notably, we observed TMPRSS12 mRNA expression in target organs of aMPV/B in chickens. Overall, our results indicate that TMPRSS12 is crucial for aMPV/B F protein proteolysis and aMPV/B infectivity and that TMPRSS12 may serve as a target for novel therapeutics and prophylactics for aMPV. Proteolysis of the aMPV F protein is a prerequisite for F protein-mediated membrane fusion of virus and cell and for aMPV infection; however, the proteases used in vitro and vivo are not clear. A combination of analyses, including overexpression, knockdown, and mutation methods, demonstrated that the transmembrane serine protease TMPRSS12 facilitated cleavage of subtype B aMPV (aMPV/B) F protein. Importantly, we located the motif in the aMPV/B F protein recognized by TMPRSS12 and the catalytic triad in TMPRSS12 that facilitated proteolysis of the aMPV/B F protein. This is the first report on TMPRSS12 as a protease for proteolysis of viral envelope

  4. A Sequence and Structure Based Method to Predict Putative Substrates, Functions and Regulatory Networks of Endo Proteases

    Science.gov (United States)

    Venkatraman, Prasanna; Balakrishnan, Satish; Rao, Shashidhar; Hooda, Yogesh; Pol, Suyog

    2009-01-01

    Background Proteases play a central role in cellular homeostasis and are responsible for the spatio- temporal regulation of function. Many putative proteases have been recently identified through genomic approaches, leading to a surge in global profiling attempts to characterize their function. Through such efforts and others it has become evident that many proteases play non-traditional roles. Accordingly, the number and the variety of the substrate repertoire of proteases are expected to be much larger than previously assumed. In line with such global profiling attempts, we present here a method for the prediction of natural substrates of endo proteases (human proteases used as an example) by employing short peptide sequences as specificity determinants. Methodology/Principal Findings Our method incorporates specificity determinants unique to individual enzymes and physiologically relevant dual filters namely, solvent accessible surface area-a parameter dependent on protein three-dimensional structure and subcellular localization. By incorporating such hitherto unused principles in prediction methods, a novel ligand docking strategy to mimic substrate binding at the active site of the enzyme, and GO functions, we identify and perform subjective validation on putative substrates of matriptase and highlight new functions of the enzyme. Using relative solvent accessibility to rank order we show how new protease regulatory networks and enzyme cascades can be created. Conclusion We believe that our physiologically relevant computational approach would be a very useful complementary method in the current day attempts to profile proteases (endo proteases in particular) and their substrates. In addition, by using functional annotations, we have demonstrated how normal and unknown functions of a protease can be envisaged. We have developed a network which can be integrated to create a proteolytic world. This network can in turn be extended to integrate other regulatory

  5. Carbohydrate protease conjugates: Stabilized proteases for peptide synthesis

    Energy Technology Data Exchange (ETDEWEB)

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

    1995-12-31

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

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

    DEFF Research Database (Denmark)

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

    1987-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    1987-08-01

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

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

    International Nuclear Information System (INIS)

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

    1987-01-01

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

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

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  10. STABILITY OF BINARY COMPLEXES OF L-ASPARTIC ACID IN ...

    African Journals Online (AJOL)

    Preferred Customer

    KEY WORDS: Binary complexes, Stability constants, Aspartic acid, Speciation, Dioxan. INTRODUCTION. 1,4-Dioxan (Dox) is ... It is miscible with water, oils, and most organic solvents, including aromatic .... of mineral acid in metal ion and ligand solutions was determined using the Gran plot method. [28, 29]. To assess the ...

  11. Production of aspartic peptidases by Aspergillus spp. using tuna ...

    African Journals Online (AJOL)

    The production of extracellular aspartic peptidase by the fungi Aspergillus niger and Aspergillus awamori was carried out in a shake flask and in stirred tank submerged fermentations using tuna cooked wastewater, an industrial effluent, as nitrogen source for culture medium. In stirred tank fermentation, biomass production ...

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

    KAUST Repository

    Li, Zhao

    2018-02-16

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

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

    KAUST Repository

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

    2018-01-01

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

  14. Monocytes can be induced by lipopolysaccharide-triggered T lymphocytes to express functional factor VII/VIIa protease activity

    OpenAIRE

    1984-01-01

    In the present study we demonstrate that human monocytes can be induced by the model stimulus, lipopolysaccharide (LPS), to produce and assemble on their surface functional Factor VII/VIIa. This protease was not induced in relatively purified monocytes alone following exposure to LPS; but was induced in the presence of Leu-3a positive helper/inducer T cells. The Factor VII/VIIa protease activity represented 35-40% of the potential initiating activity for the extrinsic coagulation pathway and ...

  15. Apical serine protease activity is necessary for assembly of a high-resistance renal collecting duct epithelium

    DEFF Research Database (Denmark)

    Steensgaard, Mette; Svenningsen, Per; Tinning, Anne R

    2010-01-01

    Abstract AIM: We hypothesized that the serine protease prostasin is necessary for differentiation of a high resistance renal collecting duct epithelium governed by glucocorticoid. METHODS: Postnatal rat kidney and adult human kidney was used to study expression and localization of prostasin......-cadherin distribution did not change. CONCLUSION: Apical, GPI-anchored, lipid raft-associated serine protease activity, compatible with prostasin, is necessary for development of a high-resistance collecting duct epithelium....

  16. Poly(Aspartic Acid) Degradation by a Sphingomonas sp. Isolated from Freshwater

    OpenAIRE

    Tabata, Kenji; Kasuya, Ken-Ichi; Abe, Hideki; Masuda, Kozue; Doi, Yoshiharu

    1999-01-01

    A poly(aspartic acid) degrading bacterium (strain KT-1 [JCM10459]) was isolated from river water and identified as a member of the genus Sphingomonas. The isolate degraded only poly(aspartic acid)s of low molecular masses (

  17. Synthesis of 6-Phosphofructose Aspartic Acid and Some Related Amadori Compounds

    OpenAIRE

    Hansen, Alexandar L.; Behrman, Edward J.

    2016-01-01

    We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid.

  18. Electron cryomicroscopy structure of a membrane-anchored mitochondrial AAA protease.

    Science.gov (United States)

    Lee, Sukyeong; Augustin, Steffen; Tatsuta, Takashi; Gerdes, Florian; Langer, Thomas; Tsai, Francis T F

    2011-02-11

    FtsH-related AAA proteases are conserved membrane-anchored, ATP-dependent molecular machines, which mediate the processing and turnover of soluble and membrane-embedded proteins in eubacteria, mitochondria, and chloroplasts. Homo- and hetero-oligomeric proteolytic complexes exist, which are composed of homologous subunits harboring an ATPase domain of the AAA family and an H41 metallopeptidase domain. Mutations in subunits of mitochondrial m-AAA proteases have been associated with different neurodegenerative disorders in human, raising questions on the functional differences between homo- and hetero-oligomeric AAA proteases. Here, we have analyzed the hetero-oligomeric yeast m-AAA protease composed of homologous Yta10 and Yta12 subunits. We combined genetic and structural approaches to define the molecular determinants for oligomer assembly and to assess functional similarities between Yta10 and Yta12. We demonstrate that replacement of only two amino acid residues within the metallopeptidase domain of Yta12 allows its assembly into homo-oligomeric complexes. To provide a molecular explanation, we determined the 12 Å resolution structure of the intact yeast m-AAA protease with its transmembrane domains by electron cryomicroscopy (cryo-EM) and atomic structure fitting. The full-length m-AAA protease has a bipartite structure and is a hexamer in solution. We found that residues in Yta12, which facilitate homo-oligomerization when mutated, are located at the interface between neighboring protomers in the hexamer ring. Notably, the transmembrane and intermembrane space domains are separated from the main body, creating a passage on the matrix side, which is wide enough to accommodate unfolded but not folded polypeptides. These results suggest a mechanism regarding how proteins are recognized and degraded by m-AAA proteases.

  19. Proteases secreted by Gnathostoma binucleatum degrade fibronectin and antibodies from mammals

    Directory of Open Access Journals (Sweden)

    Vibanco-Pérez N.

    2015-02-01

    Full Text Available Gnathostomiasis is a prevalent zoonosis in humans in some regions of the world. The genus Gnathostoma is considered an accidental parasite for humans; G. binucleatum is the endemic species in Nayarit, Mexico. This work was designed to determine the proteolytic activity of the excretory-secretory products (ESP of advanced third-stage larvae (ADVL3 of Gnathostoma binucleatum against human fibronectin and antibodies from human and sheep. Our findings showed protease activity against human fibronectin as well as sheep and human gamma globulins of the ESP at molecular weights of 80 and 56 kDa. The proteases found in the ESP of G. binucleatum are thus candidate molecules for consideration as pathogenic elements, owing to the fact that they destroy proteins of the host tissue, which probably allows them to migrate through those tissues and degrade molecules involved in the humoral immune response.

  20. The aspartic proteinase family of three Phytophthora species

    Science.gov (United States)

    2011-01-01

    Background Phytophthora species are oomycete plant pathogens with such major social and economic impact that genome sequences have been determined for Phytophthora infestans, P. sojae and P. ramorum. Pepsin-like aspartic proteinases (APs) are produced in a wide variety of species (from bacteria to humans) and contain conserved motifs and landmark residues. APs fulfil critical roles in infectious organisms and their host cells. Annotation of Phytophthora APs would provide invaluable information for studies into their roles in the physiology of Phytophthora species and interactions with their hosts. Results Genomes of Phytophthora infestans, P. sojae and P. ramorum contain 11-12 genes encoding APs. Nine of the original gene models in the P. infestans database and several in P. sojae and P. ramorum (three and four, respectively) were erroneous. Gene models were corrected on the basis of EST data, consistent positioning of introns between orthologues and conservation of hallmark motifs. Phylogenetic analysis resolved the Phytophthora APs into 5 clades. Of the 12 sub-families, several contained an unconventional architecture, as they either lacked a signal peptide or a propart region. Remarkably, almost all APs are predicted to be membrane-bound. Conclusions One of the twelve Phytophthora APs is an unprecedented fusion protein with a putative G-protein coupled receptor as the C-terminal partner. The others appear to be related to well-documented enzymes from other species, including a vacuolar enzyme that is encoded in every fungal genome sequenced to date. Unexpectedly, however, the oomycetes were found to have both active and probably-inactive forms of an AP similar to vertebrate BACE, the enzyme responsible for initiating the processing cascade that generates the Aβ peptide central to Alzheimer's Disease. The oomycetes also encode enzymes similar to plasmepsin V, a membrane-bound AP that cleaves effector proteins of the malaria parasite Plasmodium falciparum during

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

    Science.gov (United States)

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

    2017-11-29

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

  2. Cysteine proteases of pathogenic organisms

    National Research Council Canada - National Science Library

    Robinson, Mark W; Dalton, J. P

    2011-01-01

    .... Written by leading researchers from Europe, Australia and North America, this book is essential reading for students and professionals interested in human medicine and infectious disease research...

  3. Serine protease inhibitors of parasitic helminths.

    Science.gov (United States)

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

    2012-05-01

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

  4. Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1

    DEFF Research Database (Denmark)

    Rasmussen, J.G.; Riis, Simone Elkjær; Frøbert, O.

    2012-01-01

    Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible fact...

  5. Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

    OpenAIRE

    Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

    2010-01-01

    Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-tra...

  6. Inhibition of protease-inhibitor resistant hepatitis C virus replicons and infectious virus by intracellular intrabodies

    Science.gov (United States)

    Gal-Tanamy, Meital; Zemel, Romy; Bachmatov, Larissa; Jangra, Rohit K.; Shapira, Assaf; Villanueva, Rodrigo; Yi, MinKyung; Lemon, Stanley M.; Benhar, Itai; Tur-Kaspa, Ran

    2015-01-01

    Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons resistant to small molecule inhibitors of the NS3/4A protease, and replicons derived from different HCV genotypes. The combination of intrabodies and interferon-α had an additive inhibitory effect on RNA replication in the replicon model. Intrabody expression also inhibited production of infectious HCV in a cell culture system. The NS3 protease activity was inhibited by the intrabodies in NS3-expressing cells. In contrast, cell-free synthesis of HCV RNA by preformed replicase complexes was not inhibited by intrabodies, suggesting that the major mode of inhibition of viral replication is inhibition of NS3/4A protease activity and subsequent suppression of viral polyprotein processing. PMID:20705106

  7. Mitochondrial AAA proteases--towards a molecular understanding of membrane-bound proteolytic machines.

    Science.gov (United States)

    Gerdes, Florian; Tatsuta, Takashi; Langer, Thomas

    2012-01-01

    Mitochondrial AAA proteases play an important role in the maintenance of mitochondrial proteostasis. They regulate and promote biogenesis of mitochondrial proteins by acting as processing enzymes and ensuring the selective turnover of misfolded proteins. Impairment of AAA proteases causes pleiotropic defects in various organisms including neurodegeneration in humans. AAA proteases comprise ring-like hexameric complexes in the mitochondrial inner membrane and are functionally conserved from yeast to man, but variations are evident in the subunit composition of orthologous enzymes. Recent structural and biochemical studies revealed how AAA proteases degrade their substrates in an ATP dependent manner. Intersubunit coordination of the ATP hydrolysis leads to an ordered ATP hydrolysis within the AAA ring, which ensures efficient substrate dislocation from the membrane and translocation to the proteolytic chamber. In this review, we summarize recent findings on the molecular mechanisms underlying the versatile functions of mitochondrial AAA proteases and their relevance to those of the other AAA+ machines. Copyright © 2011 Elsevier B.V. All rights reserved.

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

    African Journals Online (AJOL)

    Tuoyo Aghomotsegin

    2016-11-23

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

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

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

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

    Science.gov (United States)

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

    2008-01-01

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

  12. Cysteine proteases as potential antigens in antiparasitic DNA vaccines

    DEFF Research Database (Denmark)

    Jørgensen, Louise von Gersdorff; Buchmann, Kurt

    2011-01-01

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

  13. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  14. New aspartic proteinase of Ulysses retrotransposon from Drosophila virilis.

    Science.gov (United States)

    Volkov, D A; Dergousova, N I; Rumsh, L D

    2004-06-01

    This work is focused on the investigation of a proteinase of Ulysses mobile genetic element from Drosophila virilis. The primary structure of this proteinase is suggested based on comparative analysis of amino acid sequences of aspartic proteinases from retroviruses and retrotransposons. The corresponding cDNA fragment has been cloned and expressed in E. coli. The protein accumulated in inclusion bodies. The recombinant protein (12 kD) was subjected to refolding and purified by affinity chromatography on pepstatin-agarose. Proteolytic activity of the protein was determined using oligopeptide substrates melittin and insulin B-chain. It was found that the maximum of the proteolytic activity is displayed at pH 5.5 as for the majority of aspartic proteinases. We observed that hydrolysis of B-chain of insulin was totally inhibited by pepstatin A in the micromolar concentration range. The molecular weight of the monomer of the Ulysses proteinase was determined by MALDI-TOF mass-spectrometry.

  15. Detection of Aspartic Proteinase Activities Using Gel Zymography.

    Science.gov (United States)

    Perera, Handunge Kumudu Irani

    2017-01-01

    Gel zymography is a two-stage process where the proteins from the test sample are first separated by electrophoresis followed by the detection of the activity of hydrolytic enzymes. Many zymography procedures use sodium dodecyl sulfate (SDS) polyacrylamide gels copolymerized with an appropriate substrate. The procedure described here uses native polyacrylamide gel electrophoresis (PAGE) in the absence of both SDS and substrate. In order to visualize aspartic proteinase activity, the gel is impregnated in bovine hemoglobin at pH 3.0 for 15 min after the electrophoresis procedure. Subsequently, the gel is incubated in a humid container in the absence of hemoglobin for 1 h at 37 °C. At the end, the gel is stained with amido black and destained. Clear areas against a dark background corresponding to aspartic proteinase activities can be detected.

  16. Crosslinked Aspartic Acids as Helix-Nucleating Templates.

    Science.gov (United States)

    Zhao, Hui; Liu, Qi-Song; Geng, Hao; Tian, Yuan; Cheng, Min; Jiang, Yan-Hong; Xie, Ming-Sheng; Niu, Xiao-Gang; Jiang, Fan; Zhang, Ya-Ou; Lao, Yuan-Zhi; Wu, Yun-Dong; Xu, Nai-Han; Li, Zi-Gang

    2016-09-19

    Described is a facile helix-nucleating template based on a tethered aspartic acid at the N-terminus [terminal aspartic acid (TD)]. The nucleating effect of the template is subtly influenced by the substituent at the end of the side-chain-end tether as indicated by circular dichroism, nuclear magnetic resonance, and molecular dynamics simulations. Unlike most nucleating strategies, the N-terminal amine is preserved, thus enabling further modification. Peptidomimetic estrogen receptor modulators (PERMs) constructed using this strategy show improved therapeutic properties. The current strategy can be regarded as a good complement to existing helix-stabilizing methods. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Bioproduction of L-Aspartic Acid and Cinnamic Acid by L-Aspartate Ammonia Lyase from Pseudomonas aeruginosa PAO1.

    Science.gov (United States)

    Patel, Arti T; Akhani, Rekha C; Patel, Manisha J; Dedania, Samir R; Patel, Darshan H

    2017-06-01

    Aspartase (L-aspartate ammonia lyase, EC 4.3.1.1) catalyses the reversible amination and deamination of L-aspartic acid to fumaric acid which can be used to produce important biochemical. In this study, we have explored the characteristics of aspartase from Pseudomonas aeruginosa PAO1 (PA-AspA). To overproduce PA-AspA, the 1425-bp gene was introduced in Escherichia coli BL21 and purified. A 51.0-kDa protein was observed as a homogenous purified protein on SDS-PAGE. The enzyme was optimally active at pH 8.0 and 35 °C. PA-AspA has retained 56% activity after 7 days of incubation at 35 °C, which displays the hyperthermostablility characteristics of the enzyme. PA-AspA is activated in the presence of metal ions and Mg2+ is found to be most effective. Among the substrates tested for specificity of PA-AspA, L-phenylalanine (38.35 ± 2.68) showed the highest specific activity followed by L-aspartic acid (31.21 ± 3.31) and fumarate (5.42 ± 2.94). K m values for L-phenylalanine, L-aspartic acid and fumarate were 1.71 mM, 0.346 μM and 2 M, respectively. The catalytic efficiency (k cat /K m ) for L-aspartic acid (14.18 s -1  mM -1 ) was higher than that for L-phenylalanine (4.65 s -1  mM -1 ). For bioconversion, from an initial concentration of 1000 mM of fumarate and 30 mM of L-phenylalanine, PA-AspA was found to convert 395.31 μM L-aspartic acid and 3.47 mM cinnamic acid, respectively.

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

    OpenAIRE

    Yusriah, Yusriah; Kuswytasari, Nengah Dwianita

    2013-01-01

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

  19. High throughput in vivo protease inhibitor selection platform

    DEFF Research Database (Denmark)

    2017-01-01

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

  20. Novel IgG-Degrading Enzymes of the IgdE Protease Family Link Substrate Specificity to Host Tropism of Streptococcus Species.

    Science.gov (United States)

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

    2016-01-01

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

  1. Lipase and protease extraction from activated sludge

    DEFF Research Database (Denmark)

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

    2003-01-01

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

  2. HIV-1 protease-induced apoptosis

    Czech Academy of Sciences Publication Activity Database

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

    2014-01-01

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

  3. Bacterial proteases: targets for diagnostics and therapy

    NARCIS (Netherlands)

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

    2014-01-01

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

  4. Novel peptide-based protease inhibitors

    DEFF Research Database (Denmark)

    Roodbeen, Renée

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

  5. Propagation of biochirality: crossovers and nonclassical crystallization kinetics of aspartic acid in water.

    Science.gov (United States)

    Lee, Tu; Lin, Yu Kun; Tsai, Ya Chung; Lee, Hung Lin

    2013-11-01

    All experimental procedures discussed could be treated as a screening tool for probing the existence of molecular association among the chiral molecules and the solvent system. The molecular association phases of a racemic conglomerate solution (CS) and a racemic compound solution (RCS), and the templating effect of aspartic acid solid surface were observed to minimize the chance of redissolving racemic conglomerate and racemic compound aspartic acid in water and reforming an RCS in crossovers experiments. Only 1 %wt% of l-aspartic acid was adequate enough to induce a transformation from a racemic compound aspartic acid to a racemic conglomerate aspartic acid. This would make the propagation of biochirality more feasible and sound. However, tetrapeptide, (l-aspartic acid)4 , failed to induce enantioseparation as templates purely by crystallization. Nonclassical crystallization theory was needed to take into account the existence of a CS. Fundamental parameters of the crystallization kinetics such as the induction time, interfacial energy, Gibbs energetic barrier, nucleation rate, and critical size of stable nuclei of: (i) racemic compound aspartic acid, (ii) racemic compound aspartic acid seeded with 1 %wt% l-aspartic acid, (iii) racemic conglomerate aspartic acid, and (iv) l-aspartic acid were evaluated and compared with different initial supersaturation ratios. Morphological studies of crystals grown from the crystallization kinetics were also carried out. © 2013 Wiley Periodicals, Inc.

  6. Aspartic acid racemisation in purified elastin from arteries as basis for age estimation.

    Science.gov (United States)

    Dobberstein, R C; Tung, S-M; Ritz-Timme, S

    2010-07-01

    Aspartic acid racemisation (AAR) results in an age-dependent accumulation of D: -aspartic acid in durable human proteins and can be used as a basis for age estimation. Routinely, age estimation based on AAR is performed by analysis of dentine. However, in forensic practise, teeth are not always available. Non-dental tissues for age estimation may be suitable for age estimation based on AAR if they contain durable proteins that can be purified and analysed. Elastin is such a durable protein. To clarify if purified elastin from arteries is a suitable sample for biochemical age estimation, AAR was determined in purified elastin from arteries from individuals of known age (n = 68 individuals, including n = 15 putrefied corpses), considering the influence of different stages of atherosclerosis and putrefaction on the AAR values. AAR was found to increase with age. The relationship between AAR and age was good enough to serve as basis for age estimation, but worse than known from dentinal proteins. Intravital and post-mortem degradation of elastin may have a moderate effect on the AAR values. Age estimation based on AAR in purified elastin from arteries may be a valuable additional tool in the identification of unidentified cadavers, especially in cases where other methods cannot be applied (e.g., no available teeth and body parts).

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

    International Nuclear Information System (INIS)

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

    2009-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2009-06-05

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

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

    Science.gov (United States)

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

    2015-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Luca Musante

    2015-01-01

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

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

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

    Science.gov (United States)

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

    1999-07-01

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

  13. Comparison of a soluble co-formulation of insulin degludec/insulin aspart vs biphasic insulin aspart 30 in type 2 diabetes

    DEFF Research Database (Denmark)

    Niskanen, Leo; Leiter, Lawrence A; Franek, Edward

    2012-01-01

    Insulin degludec/insulin aspart (IDegAsp) is a soluble co-formulation of insulin degludec (70%) and insulin aspart (IAsp: 30%). Here, we compare the efficacy and safety of IDegAsp, an alternative IDegAsp formulation (AF: containing 45% IAsp), and biphasic IAsp 30 (BIAsp 30)....

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

    Directory of Open Access Journals (Sweden)

    Kanupriya Miglani Sharma

    2017-06-01

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

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

    Science.gov (United States)

    Thakur, Rupamoni; Mukherjee, Ashis K

    2017-06-01

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

  16. Transcriptional and proteomic analysis of the Aspergillus fumigatus ΔprtT protease-deficient mutant.

    Science.gov (United States)

    Hagag, Shelly; Kubitschek-Barreira, Paula; Neves, Gabriela W P; Amar, David; Nierman, William; Shalit, Itamar; Shamir, Ron; Lopes-Bezerra, Leila; Osherov, Nir

    2012-01-01

    Aspergillus fumigatus is the most common opportunistic mold pathogen of humans, infecting immunocompromised patients. The fungus invades the lungs and other organs, causing severe damage. Penetration of the pulmonary epithelium is a key step in the infectious process. A. fumigatus produces extracellular proteases to degrade the host structural barriers. The A. fumigatus transcription factor PrtT controls the expression of multiple secreted proteases. PrtT shows similarity to the fungal Gal4-type Zn(2)-Cys(6) DNA-binding domain of several transcription factors. In this work, we further investigate the function of this transcription factor by performing a transcriptional and a proteomic analysis of the ΔprtT mutant. Unexpectedly, microarray analysis revealed that in addition to the expected decrease in protease expression, expression of genes involved in iron uptake and ergosterol synthesis was dramatically decreased in the ΔprtT mutant. A second finding of interest is that deletion of prtT resulted in the upregulation of four secondary metabolite clusters, including genes for the biosynthesis of toxic pseurotin A. Proteomic analysis identified reduced levels of three secreted proteases (ALP1 protease, TppA, AFUA_2G01250) and increased levels of three secreted polysaccharide-degrading enzymes in the ΔprtT mutant possibly in response to its inability to derive sufficient nourishment from protein breakdown. This report highlights the complexity of gene regulation by PrtT, and suggests a potential novel link between the regulation of protease secretion and the control of iron uptake, ergosterol biosynthesis and secondary metabolite production in A. fumigatus.

  17. Origins of hydration differences in homochiral and racemic crystals of aspartic acid.

    Science.gov (United States)

    Juliano, Thomas R; Korter, Timothy M

    2015-02-26

    The propensity for crystalline hydrates of organic molecules to form is related to the strength of the interactions between molecules, including the chiral composition of the molecular solids. Specifically, homochiral versus racemic crystalline samples can exhibit distinct differences in their ability to form energetically stable hydrates. The focus of the current study is a comparison of the crystal structures and intermolecular forces found in solid-state L-aspartic acid, DL-aspartic acid, and L-aspartic acid monohydrate. The absence of experimental evidence for the DL-aspartic acid monohydrate is considered here in terms of the enhanced thermodynamic stability of the DL-aspartic acid anhydrate crystal as compared to the L-aspartic acid anhydrate as revealed through solid-state density functional theory calculations and terahertz spectroscopic measurements. The results indicate that anhydrous DL-aspartic acid is the more stable solid, not due to intermolecular forces alone but also due to the improved conformations of the molecules within the racemic solid. Hemihydrated and monohydrated forms of DL-aspartic acid have been computationally evaluated, and in each case, the hydrates produce destabilized aspartic acid conformations that prevent DL-aspartic acid hydrate formation from occurring.

  18. Structure and mechanisms of Escherichia coli aspartate transcarbamoylase.

    Science.gov (United States)

    Lipscomb, William N; Kantrowitz, Evan R

    2012-03-20

    Enzymes catalyze a particular reaction in cells, but only a few control the rate of this reaction and the metabolic pathway that follows. One specific mechanism for such enzymatic control of a metabolic pathway involves molecular feedback, whereby a metabolite further down the pathway acts at a unique site on the control enzyme to alter its activity allosterically. This regulation may be positive or negative (or both), depending upon the particular system. Another method of enzymatic control involves the cooperative binding of the substrate, which allows a large change in enzyme activity to emanate from only a small change in substrate concentration. Allosteric regulation and homotropic cooperativity are often known to involve significant conformational changes in the structure of the protein. Escherichia coli aspartate transcarbamoylase (ATCase) is the textbook example of an enzyme that regulates a metabolic pathway, namely, pyrimidine nucleotide biosynthesis, by feedback control and by the cooperative binding of the substrate, L-aspartate. The catalytic and regulatory mechanisms of this enzyme have been extensively studied. A series of X-ray crystal structures of the enzyme in the presence and absence of substrates, products, and analogues have provided details, at the molecular level, of the conformational changes that the enzyme undergoes as it shifts between its low-activity, low-affinity form (T state) to its high-activity, high-affinity form (R state). These structural data provide insights into not only how this enzyme catalyzes the reaction between l-aspartate and carbamoyl phosphate to form N-carbamoyl-L-aspartate and inorganic phosphate, but also how the allosteric effectors modulate this activity. In this Account, we summarize studies on the structure of the enzyme and describe how these structural data provide insights into the catalytic and regulatory mechanisms of the enzyme. The ATCase-catalyzed reaction is regulated by nucleotide binding some 60

  19. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

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

  20. Comparative analysis of procoagulant and fibrinogenolytic activity of crude protease fractions of turmeric species.

    Science.gov (United States)

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

    2015-08-22

    Turmeric rhizome is a traditional herbal medicine, which has been widely used as a remedy to stop bleeding on fresh cuts and for wound healing by the rural and tribal population of India. To validate scientific and therapeutic application of turmeric rhizomes to stop bleeding on fresh cuts and its role in wound healing process. The water extracts of thoroughly scrubbed and washed turmeric rhizomes viz., Curcuma aromatica Salisb., Curcuma longa L., Curcuma caesia Roxb., Curcuma amada Roxb. and Curcuma zedoria (Christm.) Roscoe. were subjected to salting out and dialysis. The dialyzed crude enzyme fractions (CEFs) were assessed for proteolytic activity using casein as substrate and were also confirmed by caseinolytic zymography. Its coagulant activity and fibrinogenolytic activity were assessed using human citrated plasma and fibrinogen, respectively. The type of protease(s) in CEFs was confirmed by inhibition studies using specific protease inhibitors. The CEFs of C. aromatica, C. longa and C. caesia showed 1.89, 1.21 and 1.07 folds higher proteolytic activity, respectively, compared to papain. In contrast to these, C. amada and C. zedoria exhibited moderate proteolytic activity. CEFs showed low proteolytic activities compared to trypsin. The proteolytic activities of CEFs were confirmed by caseinolytic zymography. The CEFs of C. aromatica, C. longa and C. caesia showed complete hydrolysis of Aα, Bβ and γ subunits of human fibrinogen, while C. amada and C. zedoria showed partial hydrolysis. The CEFs viz., C. aromatica, C. longa, C. caesia, C. amada and C. zedoria exhibited strong procoagulant activity by reducing the human plasma clotting time from 172s (Control) to 66s, 84s 88s, 78s and 90s, respectively. The proteolytic activity of C. aromatica, C. longa, C. caesia and C. amada was inhibited (>82%) by PMSF, suggesting the possible presence of a serine protease(s). However, C. zedoria showed significant inhibition (60%) against IAA and moderate inhibition (30

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

    International Nuclear Information System (INIS)

    Yedidi, Ravikiran S.; Muhuhi, Joseck M.; Liu, Zhigang; Bencze, Krisztina Z.; Koupparis, Kyriacos; O’Connor, Carrie E.; Kovari, Iulia A.; Spaller, Mark R.; Kovari, Ladislau C.

    2013-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-09-06

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

  3. Metabolism of [14C] bicarbonate by Streptococcus lactis: the synthesis, uptake and excretion of aspartate by resting cells

    International Nuclear Information System (INIS)

    Hillier, A.J.; Rice, G.H.; Jago, G.R.

    1978-01-01

    Resting cells of Streptococcus lactis C10 were able to synthesize aspartic acid de novo but could not actively transport aspartic acid into the cell. Intracellular aspartate was excreted from the cell in the presence of glucose but did not exchange with any extracellular amino acids. The results indicate that Str. lactis C10 obtains the aspartic acid it requires for growth by bicarbonate fixation instead of by the utilization of extracellular aspartic acid. (author)

  4. Luminometric method for screening retroviral protease inhibitors

    Czech Academy of Sciences Publication Activity Database

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

    2005-01-01

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

  5. Role of Proteases in Chronic Obstructive Pulmonary Disease

    Directory of Open Access Journals (Sweden)

    Kailash C. Pandey

    2017-08-01

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

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

    Directory of Open Access Journals (Sweden)

    R. Satheeskumar

    2013-10-01

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

  7. Understanding serine proteases implications on Leishmania spp lifecycle.

    Science.gov (United States)

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

    2018-01-01

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

  8. Aspartate and glutamate mimetic structures in biologically active compounds.

    Science.gov (United States)

    Stefanic, Peter; Dolenc, Marija Sollner

    2004-04-01

    Glutamate and aspartate are frequently recognized as key structural elements for the biological activity of natural peptides and synthetic compounds. The acidic side-chain functionality of both the amino acids provides the basis for the ionic interaction and subsequent molecular recognition by specific receptor sites that results in the regulation of physiological or pathophysiological processes in the organism. In the development of new biologically active compounds that possess the ability to modulate these processes, compounds offering the same type of interactions are being designed. Thus, using a peptidomimetic design approach, glutamate and aspartate mimetics are incorporated into the structure of final biologically active compounds. This review covers different bioisosteric replacements of carboxylic acid alone, as well as mimetics of the whole amino acid structure. Amino acid analogs presented include those with different distances between anionic moieties, and analogs with additional functional groups that result in conformational restriction or alternative interaction sites. The article also provides an overview of different cyclic structures, including various cycloalkane, bicyclic and heterocyclic analogs, that lead to conformational restriction. Higher di- and tripeptide mimetics in which carboxylic acid functionality is incorporated into larger molecules are also reviewed. In addition to the mimetic structures presented, emphasis in this article is placed on their steric and electronic properties. These mimetics constitute a useful pool of fragments in the design of new biologically active compounds, particularly in the field of RGD mimetics and excitatory amino acid agonists and antagonists.

  9. Molecular adaptation of a plant-bacterium outer membrane protease towards plague virulence factor Pla

    Science.gov (United States)

    2011-01-01

    Background Omptins are a family of outer membrane proteases that have spread by horizontal gene transfer in Gram-negative bacteria that infect vertebrates or plants. Despite structural similarity, the molecular functions of omptins differ in a manner that reflects the life style of their host bacteria. To simulate the molecular adaptation of omptins, we applied site-specific mutagenesis to make Epo of the plant pathogenic Erwinia pyrifoliae exhibit virulence-associated functions of its close homolog, the plasminogen activator Pla of Yersinia pestis. We addressed three virulence-associated functions exhibited by Pla, i.e., proteolytic activation of plasminogen, proteolytic degradation of serine protease inhibitors, and invasion into human cells. Results Pla and Epo expressed in Escherichia coli are both functional endopeptidases and cleave human serine protease inhibitors, but Epo failed to activate plasminogen and to mediate invasion into a human endothelial-like cell line. Swapping of ten amino acid residues at two surface loops of Pla and Epo introduced plasminogen activation capacity in Epo and inactivated the function in Pla. We also compared the structure of Pla and the modeled structure of Epo to analyze the structural variations that could rationalize the different proteolytic activities. Epo-expressing bacteria managed to invade human cells only after all extramembranous residues that differ between Pla and Epo and the first transmembrane β-strand had been changed. Conclusions We describe molecular adaptation of a protease from an environmental setting towards a virulence factor detrimental for humans. Our results stress the evolvability of bacterial β-barrel surface structures and the environment as a source of progenitor virulence molecules of human pathogens. PMID:21310089

  10. Monitoring single protease activities on triple-helical collagen molecules

    Science.gov (United States)

    Harzar, Raj; Froberg, James; Srivastava, D. K.; Choi, Yongki

    Matrix metalloproteinases (MMPs), a particular family of proteases, play a pivotal role in degrading the extracellular matrix (ECM). It has been known for more than 40 years that MMPs are closely involved in multiple human cancers during cell growth, invasion, and metastasis. However, the mechanisms of MMP activity are far from being understood. Here, we monitored enzymatic processing of MMPs with two complementary approaches, atomic force microscopy and nanocircuits measurements. AFM measurements demonstrated that incubation of collagen monomers with MMPs resulted in a single position cleavage, producing 3/4 and 1/4 collagen fragments. From electronic monitoring of single MMP nanocircuit measurements, we were able to capture a single cleavage event with a rate of 0.012 Hz, which were in good agreement with fluorescence assay measurements. This work was supported financially by the NIGMS/NIH (P30GM103332-02) and ND NASA EPSCoR RID Grant.

  11. Extracellular Protease Activity of Enteropathogenic Escherechia coli on Mucin Substrate

    Directory of Open Access Journals (Sweden)

    SRI BUDIARTI

    2007-03-01

    Full Text Available Enteropathogenic Escherichia coli (EPEC causes gastrointestinal infections in human. EPEC invasion was initiated by attachment and aggressive colonization on intestinal surface. Attachment of EPEC alter the intestine mucosal cells. Despite this, the pathogenic mechanism of EPEC infectior has not been fully understood. This research hypothesizes that extracellular proteolytic enzymes is necessary for EPEC colonization. The enzyme is secreted into gastrointestinal milieu and presumably destroy mucus layer cover the gastrointestinal tract. The objective of this study was to assay EPEC extracellular protease enzyme by using mucin substrate. The activity of EPEC extracellular proteolytic enzyme on 1% mucin substrate was investigated. Non-pathogenic E. coli was used as a negative control. Positive and tentative controls were Yersinia enterocolitica and Salmonella. Ten EPEC strains were assayed, seven of them were able to degrade mucin, and the highest activity was produced by K1.1 strain. Both positive and tentative controls also showed the ability to digest 0.20% mucin.

  12. Interaction Between Some Monosaccharides and Aspartic Acid in Dilute Aqueous Solutions

    OpenAIRE

    Kulikova, Galina A.; Parfenyuk, Elena V.

    2007-01-01

    Interaction between aspartic acid and d-glucose, d-galactose, and d-fructose has been studied by isothermal titration calorimetry, calorimetry of dissolution, and densimetry. It has been found that d-glucose and d-fructose form thermodynamically stable associates with aspartic acid, in contrast to d-galactose. The selectivity in the interaction of aspartic acid with monosaccharides is affected by their stereochemical structures.

  13. Neutrophil Protease Cleavage of Von Willebrand Factor in Glomeruli – An Anti-thrombotic Mechanism in the Kidney

    Directory of Open Access Journals (Sweden)

    Ramesh Tati

    2017-02-01

    Full Text Available Adequate cleavage of von Willebrand factor (VWF prevents formation of thrombi. ADAMTS13 is the main VWF-cleaving protease and its deficiency results in development of thrombotic microangiopathy. Besides ADAMTS13 other proteases may also possess VWF-cleaving activity, but their physiological importance in preventing thrombus formation is unknown. This study investigated if, and which, proteases could cleave VWF in the glomerulus. The content of the glomerular basement membrane (GBM was studied as a reflection of processes occurring in the subendothelial glomerular space. VWF was incubated with human GBMs and VWF cleavage was assessed by multimer structure analysis, immunoblotting and mass spectrometry. VWF was cleaved into the smallest multimers by the GBM, which contained ADAMTS13 as well as neutrophil proteases, elastase, proteinase 3 (PR3, cathepsin-G and matrix-metalloproteinase 9. The most potent components of the GBM capable of VWF cleavage were in the serine protease or metalloprotease category, but not ADAMTS13. Neutralization of neutrophil serine proteases inhibited GBM-mediated VWF-cleaving activity, demonstrating a marked contribution of elastase and/or PR3. VWF-platelet strings formed on the surface of primary glomerular endothelial cells, in a perfusion system, were cleaved by both elastase and the GBM, a process blocked by elastase inhibitor. Ultramorphological studies of the human kidney demonstrated neutrophils releasing elastase into the GBM. Neutrophil proteases may contribute to VWF cleavage within the subendothelium, adjacent to the GBM, and thus regulate thrombus size. This anti-thrombotic mechanism would protect the normal kidney during inflammation and could also explain why most patients with ADAMTS13 deficiency do not develop severe kidney failure.

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

    Directory of Open Access Journals (Sweden)

    Rajesh Prasad

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

  15. Biphasic insulin aspart 30/70 (BIAsp 30 in the treatment of type 1 and type 2 diabetes

    Directory of Open Access Journals (Sweden)

    Paul Valensi

    2009-06-01

    Full Text Available Paul ValensiDepartment of Endocrinology-Diabetology-Nutrition, Jean Verdier Hospital, AP-HP, Paris Nord University, CRNH-IdF, Bondy, FranceAbstract: The pharmacological advantages of the rapid-acting analog, insulin aspart, over human insulin have contributed to the widespread prescription of the premix, biphasic insulin aspart 30/70 (BIAsp 30, in type 1 (T1DM and type 2 diabetes (T2DM. This article reviews the available literature on the pharmacology, efficacy and safety of BIAsp 30 in T1DM and T2DM from an online search of the PubMed database. Following injection, BIAsp 30 reaches higher plasma insulin levels more quickly than human premix or basal insulin, giving effective reduction of postprandial hyperglycemia. In T1DM patients, randomized controlled trials (RCTs have shown that HbA1c reduction is similar, but postprandial glycemic control is better, with BIAsp 30 than with human insulin regimens. In T2DM patients, lowering of HbA1c and postprandial hyperglycemia with BIAsp 30 compare favorably with optimized oral antidiabetes drug treatment, insulin glargine, and, in obese patients, human premix. An increase in minor hypoglycemia with BIAsp 30 relative to basal insulin has been reported in T2DM patients, but major and nocturnal hypoglycemia rates are generally low. Findings from RCTs in T2DM patients are supported by large observational studies. In summary, BIAsp 30 once to three times daily represents a simple and effective tool for the modern management of diabetes.Keywords: biphasic insulin aspart, BIAsp 30, premix, type 1 diabetes, type 2 diabetes

  16. Towards tricking a pathogen's protease into fighting infection: the 3D structure of a stable circularly permuted onconase variant cleavedby HIV-1 protease.

    Directory of Open Access Journals (Sweden)

    Mariona Callís

    Full Text Available Onconase® is a highly cytotoxic amphibian homolog of Ribonuclease A. Here, we describe the construction of circularly permuted Onconase® variants by connecting the N- and C-termini of this enzyme with amino acid residues that are recognized and cleaved by the human immunodeficiency virus protease. Uncleaved circularly permuted Onconase® variants are unusually stable, non-cytotoxic and can internalize in human T-lymphocyte Jurkat cells. The structure, stability and dynamics of an intact and a cleaved circularly permuted Onconase® variant were determined by Nuclear Magnetic Resonance spectroscopy and provide valuable insight into the changes in catalytic efficiency caused by the cleavage. The understanding of the structural environment and the dynamics of the activation process represents a first step toward the development of more effective drugs for the treatment of diseases related to pathogens expressing a specific protease. By taking advantage of the protease's activity to initiate a cytotoxic cascade, this approach is thought to be less susceptible to known resistance mechanisms.

  17. Pediatric Opsoclonus-Myoclonus-Ataxia Syndrome Associated With Anti-N-methyl-D-aspartate Receptor Encephalitis.

    Science.gov (United States)

    Player, Brittany; Harmelink, Matthew; Bordini, Brett; Weisgerber, Michael; Girolami, Michael; Croix, Michael

    2015-11-01

    The full clinical spectrum of anti-N-methyl-D-aspartate receptor encephalitis is unknown in the pediatric population. We describe a previously healthy 4-year-old girl presenting with opsoclonus-myoclonus together with ataxia who had NR1-specific, anti-N-methyl-D-aspartate receptor antibodies in the cerebral spinal fluid. The presence of NR1-specific, anti-N-methyl-D-aspartate receptor antibodies in the setting of opsoclonus-myoclonus and ataxia syndrome may represent an expansion of the clinical presentations of anti-N-methyl-D-aspartate receptor encephalitis. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Growth and characterization of KDP crystals doped with L-aspartic acid.

    Science.gov (United States)

    Krishnamurthy, R; Rajasekaran, R; Samuel, Bincy Susan

    2013-03-01

    Potassium Dihydrogen Phosphate (KDP) doped with L-aspartic acid has been grown by solvent slow evaporation technique from a mixture of aqueous solution of KDP and 0.7% of L-aspartic acid at room temperature. The grown crystals were characterized by powder X-ray diffraction, UV-visible, FTIR analysis. The doping of aspartic acid was confirmed by FTIR spectrum. The Nonlinear optical property (SHG) of L-aspartic acid doped KDP has been confirmed. Microhardness studies were carried out on the grown crystal. Copyright © 2012 Elsevier B.V. All rights reserved.

  19. Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines.

    Science.gov (United States)

    Henry, Conor M; Sullivan, Graeme P; Clancy, Danielle M; Afonina, Inna S; Kulms, Dagmar; Martin, Seamus J

    2016-02-02

    Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ~500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines

    Directory of Open Access Journals (Sweden)

    Conor M. Henry

    2016-02-01

    Full Text Available Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ∼500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis.

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

    International Nuclear Information System (INIS)

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

    2007-01-01

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

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

    OpenAIRE

    Kyöstiö, Sirkka R. M.

    1990-01-01

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

  3. Economic Methods of Ginger Protease'sextraction and Purification

    Science.gov (United States)

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

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

  4. Nontypeable Haemophilus influenzae induces sustained lung oxidative stress and protease expression.

    Directory of Open Access Journals (Sweden)

    Paul T King

    Full Text Available Nontypeable Haemophilus influenzae (NTHi is a prevalent bacterium found in a variety of chronic respiratory diseases. The role of this bacterium in the pathogenesis of lung inflammation is not well defined. In this study we examined the effect of NTHi on two important lung inflammatory processes 1, oxidative stress and 2, protease expression. Bronchoalveolar macrophages were obtained from 121 human subjects, blood neutrophils from 15 subjects, and human-lung fibroblast and epithelial cell lines from 16 subjects. Cells were stimulated with NTHi to measure the effect on reactive oxygen species (ROS production and extracellular trap formation. We also measured the production of the oxidant, 3-nitrotyrosine (3-NT in the lungs of mice infected with this bacterium. NTHi induced widespread production of 3-NT in mouse lungs. This bacterium induced significantly increased ROS production in human fibroblasts, epithelial cells, macrophages and neutrophils; with the highest levels in the phagocytic cells. In human macrophages NTHi caused a sustained, extracellular production of ROS that increased over time. The production of ROS was associated with the formation of macrophage extracellular trap-like structures which co-expressed the protease metalloproteinase-12. The formation of the macrophage extracellular trap-like structures was markedly inhibited by the addition of DNase. In this study we have demonstrated that NTHi induces lung oxidative stress with macrophage extracellular trap formation and associated protease expression. DNase inhibited the formation of extracellular traps.

  5. Experimental evidence for a chiral symmetry-breaking mechanism in aspartic acid: Lattice and sub-lattice matching

    Science.gov (United States)

    Teschke, Omar; Soares, David Mendez

    2017-10-01

    A mother crystal formed from a transient molecular structure of (D+L) aspartic acid in solution is reported. Hexagonal structures with a lattice constant of 1.04 nm were crystallized from a solution in which three aspartic acid species coexist: right- and left-handed enantiomorphs, denoted D-aspartic and L-aspartic, respectively, and transitory (D+L) aspartic acid specie. Atomic force microscopy images of the crystalline deposits reveal domains of the transitory (D+L) aspartic acid crystal forming the substrate deposit on silicon wafers, and on top of this hexagonal lattice only L-aspartic acid is observed to conform and crystallize. A preferential crystallization mechanism is then observed for (D+L) aspartic acid crystals that seed only L-aspartic deposits by the geometrical matching of their multiple hexagonal lattice structures with periodicities of 1.04 nm and 0.52 nm, respectively.

  6. Indispensable Role of Proteases in Plant Innate Immunity.

    Science.gov (United States)

    Balakireva, Anastasia V; Zamyatnin, Andrey A

    2018-02-23

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

  7. Heterologous expression of Hordeum vulgare cysteine protease in yeast

    DEFF Research Database (Denmark)

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

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

  8. Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

    Directory of Open Access Journals (Sweden)

    Tanveer Majeed

    2013-01-01

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

  9. Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

    Directory of Open Access Journals (Sweden)

    Suk Ho Choi

    2011-09-01

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

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

    International Nuclear Information System (INIS)

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

    2011-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2002-08-01

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

  12. N-methyl-D-aspartic acid receptor agonists

    DEFF Research Database (Denmark)

    Madsen, U; Frydenvang, Karla Andrea; Ebert, B

    1996-01-01

    (R,S)-2-Amino-2-(3-hydroxy-5-methyl-4-isoxazolyl)acetic acid [(R,S)-AMAA, 4] is a potent and selective agonist at the N-methyl-D-aspartic acid (NMDA) subtype of excitatory amino acid receptors. Using the Ugi "four-component condensation" method, the two diastereomers (2R)- and (2S)-2-[3-(benzyloxy......) showed peak affinity for [3H]AMPA receptor sites (IC50 = 72 +/- 13 microM) and was shown to be a more potent inhibitor of [3H]CPP binding (IC50 = 3.7 +/- 1.5 microM) than (S)-AMAA (9) (IC50 = 61 +/- 6.4 microM). Neither enantiomer of AMAA affected [3H]kainic acid receptor binding significantly...

  13. D-aspartic acid in aged mouse skin and lens

    International Nuclear Information System (INIS)

    Fujii, Noriko; Muraoka, Shiro; Harada, Kaoru; Tamanoi, Itsuro; Joshima, Hisamasa; Kashima, Masatoshi.

    1987-01-01

    D-aspartic acid (D-Asp) was detected in the skin and lens from naturally aged mice. An analysis of the amino acid composition indicated that D-Asp did not derive from collagen. An immunological analysis using Oucterlony's agar diffusion method also confirmed that the protein containing D-Asp was not a serum protein. The process producing D-Asp is regarded as one other than racemization because the life span of mice is not long enough to permit D-Asp by racemization. Continuous low-dose-rate gamma-irradiation (37R per day) for 102 to 112 days did not increase significantly the amount of D-Asp in skin and lens of mice. (author)

  14. D-Aspartate drinking solution alleviates pain and cognitive impairment in neuropathic mice.

    Science.gov (United States)

    Palazzo, Enza; Luongo, Livio; Guida, Francesca; Marabese, Ida; Romano, Rosaria; Iannotta, Monica; Rossi, Francesca; D'Aniello, Antimo; Stella, Luigi; Marmo, Federica; Usiello, Alessandro; de Bartolomeis, Andrea; Maione, Sabatino; de Novellis, Vito

    2016-07-01

    D-Aspartate (D-Asp) is a free D-amino acid detected in multiple brain regions and putative precursor of endogenous N-methyl-D-aspartate (NMDA) acting as agonist at NMDA receptors. In this study, we investigated whether D-Asp (20 mM) in drinking solution for 1 month affects pain responses and pain-related emotional, and cognitive behaviour in a model of neuropathic pain induced by the spared nerve injury (SNI) of the sciatic nerve in mice. SNI mice developed mechanical allodynia and motor coordination impairment 30 days after SNI surgery. SNI mice showed cognitive impairment, anxiety and depression-like behaviour, reduced sociability in the three chamber sociability paradigm, increased expression of NR2B subunit of NMDA receptor and Homer 1a in the medial prefrontal cortex (mPFC). The expression of (post synaptic density) PSD-95 and Shank 1was instead unaffected in the mPFC of the SNI mice. Treatment with D-Asp drinking solution, started right after the SNI (day 0), alleviated mechanical allodynia, improved cognition and motor coordination and increased social interaction. D-Asp also restored the levels of extracellular D-Asp, Homer 1a and NR2B subunit of the NMDA receptor to physiological levels and reduced Shank1 and PSD-95 protein levels in the mPFC. Amitriptyline, a tricyclic antidepressant used also to alleviate neuropathic pain in humans, reverted mechanical allodynia and cognitive impairment, and unlike D-Asp, was effective in reducing depression and anxiety-like behaviour in the SNI mice and increased PSD protein level. Altogether these findings demonstrate that D-Asp improves sensorial, motor and cognitive-like symptoms related to chronic pain possibly through glutamate neurotransmission normalization in neuropathic mice.

  15. Reversible Unfolding of Rhomboid Intramembrane Proteases.

    Science.gov (United States)

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

    2016-03-29

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

  16. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

    Directory of Open Access Journals (Sweden)

    Evan L Pannkuk

    Full Text Available White nose syndrome (WNS is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1 was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE, broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

  17. Isolation and identification of an extracellular subtilisin-like serine protease secreted by the bat pathogen Pseudogymnoascus destructans.

    Science.gov (United States)

    Pannkuk, Evan L; Risch, Thomas S; Savary, Brett J

    2015-01-01

    White nose syndrome (WNS) is a cutaneous fungal disease of bats. WNS is responsible for unprecedented mortalities in North American cave bat populations. There have been few descriptions of enzyme activities that may function in WNS host/pathogen interactions, while no study has isolated and described secreted proteases. To address the hypothesis that Pseudogymnoascus destructans secretes extracellular proteases that function in wing necrosis during WNS infection, the object of this study was to culture P. destructans on various media, then isolate and structurally identify those proteases accumulated stably in the culture medium. We found a single dominant protease activity on minimal nutrient broth enriched with protein substrates, which was strongly inhibited by phenylmethylsulfonyl fluoride. This P. destructans serine protease (PdSP1) was isolated by preparative isoelectric focusing and concanavalin A lectin affinity chromatography. PdSP1 showed a molecular weight 27,900 (estimated by SDS-PAGE), broad pH optimum 6-8, and temperature optimum 60°C. Structural characterization of PdSP1 by MALDI-TOF MS, Orbitrap MS/MS, and Edman amino-terminal peptide sequencing matched it directly to a hypothetical protein accession from the sequenced P. destructans genome that is further identified as a MEROPS family S8A subtilisin-like serine peptidase. Two additional isoforms, PdSP2 and PdSP3, were identified in the P. destructans genome with 90% and 53% homology, respectively. P. destructans S8A serine proteases showed closer sequence conservation to P. pannorum and plant pathogenic fungi than to human pathogenic dermatophytes. Peptide-specific polyclonal antibodies developed from the PdSP1 sequence detected the protein in western blots. These subtilisin-like serine proteases are candidates for further functional studies in WNS host-pathogen interaction.

  18. Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

    Directory of Open Access Journals (Sweden)

    Seiichi Mawatari

    Full Text Available BACKGROUND: It has been hypothesized that persistent hepatitis C virus (HCV infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4, composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. METHODS: Human C4 was incubated with HCV nonstructural (NS 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. RESULTS: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. CONCLUSIONS: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

  19. Modularly Constructed Synthetic Granzyme B Molecule Enables Interrogation of Intracellular Proteases for Targeted Cytotoxicity.

    Science.gov (United States)

    Ho, Patrick; Ede, Christopher; Chen, Yvonne Y

    2017-08-18

    Targeted therapies promise to increase the safety and efficacy of treatments against diseases ranging from cancer to viral infections. However, the vast majority of targeted therapeutics relies on the recognition of extracellular biomarkers, which are rarely restricted to diseased cells and are thus prone to severe and sometimes-fatal off-target toxicities. In contrast, intracellular antigens present a diverse yet underutilized repertoire of disease markers. Here, we report a protein-based therapeutic platform-termed Cytoplasmic Oncoprotein VErifier and Response Trigger (COVERT)-which enables the interrogation of intracellular proteases to trigger targeted cytotoxicity. COVERT molecules consist of the cytotoxic protein granzyme B (GrB) fused to an inhibitory N-terminal peptide, which can be removed by researcher-specified proteases to activate GrB function. We demonstrate that fusion of a small ubiquitin-like modifier 1 (SUMO1) protein to GrB yields a SUMO-GrB molecule that is specifically activated by the cancer-associated sentrin-specific protease 1 (SENP1). SUMO-GrB selectively triggers apoptotic phenotypes in HEK293T cells that overexpress SENP1, and it is highly sensitive to different SENP1 levels across cell lines. We further demonstrate the rational design of additional COVERT molecules responsive to enterokinase (EK) and tobacco etch virus protease (TEVp), highlighting the COVERT platform's modularity and adaptability to diverse protease targets. As an initial step toward engineering COVERT-T cells for adoptive T-cell therapy, we verified that primary human T cells can express, package, traffic, and deliver engineered GrB molecules in response to antigen stimulation. Our findings set the foundation for future intracellular-antigen-responsive therapeutics that can complement surface-targeted therapies.

  20. 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,

  1. Cleavage specificity analysis of six type II transmembrane serine proteases (TTSPs using PICS with proteome-derived peptide libraries.

    Directory of Open Access Journals (Sweden)

    Olivier Barré

    Full Text Available Type II transmembrane serine proteases (TTSPs are a family of cell membrane tethered serine proteases with unclear roles as their cleavage site specificities and substrate degradomes have not been fully elucidated. Indeed just 52 cleavage sites are annotated in MEROPS, the database of proteases, their substrates and inhibitors.To profile the active site specificities of the TTSPs, we applied Proteomic Identification of protease Cleavage Sites (PICS. Human proteome-derived database searchable peptide libraries were assayed with six human TTSPs (matriptase, matriptase-2, matriptase-3, HAT, DESC and hepsin to simultaneously determine sequence preferences on the N-terminal non-prime (P and C-terminal prime (P' sides of the scissile bond. Prime-side cleavage products were isolated following biotinylation and identified by tandem mass spectrometry. The corresponding non-prime side sequences were derived from human proteome databases using bioinformatics. Sequencing of 2,405 individual cleaved peptides allowed for the development of the family consensus protease cleavage site specificity revealing a strong specificity for arginine in the P1 position and surprisingly a lysine in P1' position. TTSP cleavage between R↓K was confirmed using synthetic peptides. By parsing through known substrates and known structures of TTSP catalytic domains, and by modeling the remainder, structural explanations for this strong specificity were derived.Degradomics analysis of 2,405 cleavage sites revealed a similar and characteristic TTSP family specificity at the P1 and P1' positions for arginine and lysine in unfolded peptides. The prime side is important for cleavage specificity, thus making these proteases unusual within the tryptic-enzyme class that generally has overriding non-prime side specificity.

  2. Synthesis of 6-phosphofructose aspartic acid and some related Amadori compounds.

    Science.gov (United States)

    Hansen, Alexandar L; Behrman, Edward J

    2016-08-05

    We describe the synthesis and characterization of 6-phosphofructose-aspartic acid, an intermediate in the metabolism of fructose-asparagine by Salmonella. We also report improved syntheses of fructose-asparagine itself and of fructose-aspartic acid. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Involvement of three meningococcal surface-exposed proteins, the heparin-binding protein NhbA, the α-peptide of IgA protease and the autotransporter protease NalP, in initiation of biofilm formation

    KAUST Repository

    Arenas, Jesú s; Nijland, Reindert; Rodriguez, Francisco J.; Bosma, Tom N. P.; Tommassen, Jan

    2012-01-01

    Neisseria meningitidis is a common and usually harmless inhabitant of the mucosa of the human nasopharynx, which, in rare cases, can cross the epithelial barrier and cause meningitis and sepsis. Biofilm formation favours the colonization of the host and the subsequent carrier state. Two different strategies of biofilm formation, either dependent or independent on extracellular DNA (eDNA), have been described for meningococcal strains. Here, we demonstrate that the autotransporter protease NalP, the expression of which is phase variable, affects eDNA-dependent biofilm formation in N.meningitidis. The effect of NalP was found in biofilm formation under static and flow conditions and was dependent on its protease activity. Cleavage of the heparin-binding antigen NhbA and the α-peptide of IgA protease, resulting in the release of positively charged polypeptides from the cell surface, was responsible for the reduction in biofilm formation when NalP is expressed. Both NhbA and the α-peptide of IgA protease were shown to bind DNA. We conclude that NhbA and the α-peptide of IgA protease are implicated in biofilm formation by binding eDNA and that NalP is an important regulator of this process through the proteolysis of these surface-exposed proteins. © 2012 Blackwell Publishing Ltd.

  4. Involvement of three meningococcal surface-exposed proteins, the heparin-binding protein NhbA, the α-peptide of IgA protease and the autotransporter protease NalP, in initiation of biofilm formation

    KAUST Repository

    Arenas, Jesús

    2012-12-04

    Neisseria meningitidis is a common and usually harmless inhabitant of the mucosa of the human nasopharynx, which, in rare cases, can cross the epithelial barrier and cause meningitis and sepsis. Biofilm formation favours the colonization of the host and the subsequent carrier state. Two different strategies of biofilm formation, either dependent or independent on extracellular DNA (eDNA), have been described for meningococcal strains. Here, we demonstrate that the autotransporter protease NalP, the expression of which is phase variable, affects eDNA-dependent biofilm formation in N.meningitidis. The effect of NalP was found in biofilm formation under static and flow conditions and was dependent on its protease activity. Cleavage of the heparin-binding antigen NhbA and the α-peptide of IgA protease, resulting in the release of positively charged polypeptides from the cell surface, was responsible for the reduction in biofilm formation when NalP is expressed. Both NhbA and the α-peptide of IgA protease were shown to bind DNA. We conclude that NhbA and the α-peptide of IgA protease are implicated in biofilm formation by binding eDNA and that NalP is an important regulator of this process through the proteolysis of these surface-exposed proteins. © 2012 Blackwell Publishing Ltd.

  5. The putative effects of D-Aspartic acid on blood testosterone levels: A systematic review

    Directory of Open Access Journals (Sweden)

    Farzad Roshanzamir

    2017-08-01

    Full Text Available Background: D-Aspartic acid (D-Asp is in invertebrate and vertebrate neuroendocrine tissues, where it carries out important physiological functions. Recently, it has been reported that D-Asp is involved in the synthesis and release of testosterone and is assumed can be used as a testosterone booster for infertile men, and by athletes to increase muscle mass and strength. Objective: The aim of this review is to summarize available evidence related to the effects of D-Asp on serum testosterone levels. Materials and Methods: We conducted a systematic review of all type studies, which evaluated the effect of the D-Asp on blood testosterone including published papers until October 2015, using PubMed, ISI Web of Science, ProQuest and Scopus database. Results: With 396 retrieved records, 23 animal studies and 4 human studies were included. In vivo and in vitro animal studies revealed the effect of D-Asp depending on species, sex and organ-specific. Our results showed that exogenous D-Asp enhances testosterone levels in male animal’s studies, whereas studies in human yielded inconsistent results. The evidence for this association in man is still sparse, mostly because of limited number and poor quality studies. Conclusion: There is an urgent need for more and well-designed human clinical trials with larger sample sizes and longer duration to investigate putative effects of D-Asp on testosterone concentrations.

  6. Photosynthetic metabolism of malate and aspartate in Flaveria trinervia a C4 dicot

    International Nuclear Information System (INIS)

    Moore, B.A.

    1986-01-01

    C 4 species are known to vary in their apparent relative use of malate and aspartate to mediate carbon flux through the C 4 cycle. These studies investigate some of the adjustments in photosynthetic carbon metabolism that occur during a dark to light transition and during expansion of leaves of Flaveria trinervia, a C 4 dicot. Enzyme localization studies with isolated leaf mesophyll and bundle sheath protoplasts, indicated that both C 4 acids are formed in the mesophyll chloroplast, and that aspartate is metabolized to malate in the bundle sheath chloroplast prior to decaroxylation there. During photosynthetic induction, the partitioning of 14 CO 2 between malate and aspartate showed a single oscillation of increased aspartate labelling after 5 min of illumination. Turnover of [4-14C] (malate plus aspartate) was slow initially during illumination, prior to establishment of active pools of C 4 cycle metabolites

  7. Protection against ionising radiation and synergism with thiols by zinc aspartate

    International Nuclear Information System (INIS)

    Floersheim, G.L.; Floersheim, P.

    1986-01-01

    Pre-treatment with zinc aspartate protected mice against the lethal effects of radiation and raised the LD 50 from 8 gy to 12.2 Gy. Zinc chloride and zinc sulphate were clearly less active. The radioprotective effect of zinc aspartate was equivalent to cysteamine and slightly inferior to S,2-aminoethylisothiourea (AET). Zinc aspartate displayed a similar therapeutic index to the thiols but could be applied at an earlier time before irradiation. Synergistic effects occurred with the combined administration of zinc aspartate and thiols. By giving zinc aspartate with cysteamine, the LD 50 was increased to 13.25 Gy and, by combining it in the optimal protocol with AET, to 17.3 Gy. The radioprotection by zinc and its synergism with thiols is explained by the stabilisation of thiols through the formation of zinc complexes. (author)

  8. Strains of Lactococcus lactis with a partial pyrimidine requirement show sensitivity toward aspartic acid

    DEFF Research Database (Denmark)

    Wadskov-Hansen, Steen Lyders Lerche; Martinussen, Jan

    2009-01-01

    The growth rate of the widely used laboratory strain Lactococcus lactis subsp. cremoris LM0230 was reduced if aspartic acid were present in the growth medium. The strain LM0230 is a plasmid- and phage-cured derivative of L. lactis subsp. cremoris C2, the ancestor of the original dairy isolate L...... with the wild-type strain, and this varied with the concentration of aspartic acid. The observed effect of aspartate could be explained by the accumulation of the toxic pyrimidine de novo pathway intermediate, carbamoyl aspartate. Assays of the pyrimidine biosynthetic enzymes of L. lactis LM0230 showed...... that the partial pyrimidine requirement can be explained by a low specific activity of the pyrimidine biosynthetic enzymes. In conclusion, L. lactis LM0230 during the process of plasmid- and prophage-curing has acquired a partial pyrimidine requirement resulting in sensitivity toward aspartic acid....

  9. Scrapie susceptibility-linked polymorphisms modulate the in vitro conversion of sheep prion protein to protease-resistant forms

    NARCIS (Netherlands)

    Bossers, A.; Belt, P.B.G.M.; Raymond, G.J.; Caughey, B.; Vries, de R.; Smits, M.

    1997-01-01

    Prion diseases are natural transmissible neurodegenerative disorders in humans and animals. They are characterized by the accumulation of a protease-resistant scrapie-associated prion protein (PrPSc) of the host-encoded cellular prion protein (PrPC) mainly in the central nervous system.

  10. Extracellular protease mRNAs are predominantly expressed in the stromal areas of microdissected mouse breast carcinomas

    DEFF Research Database (Denmark)

    Pedersen, Tanja Xenia; Pennington, Caroline J; Almholt, Kasper

    2005-01-01

    Solid tumors synthesize a number of extracellular matrix-degrading proteases that are important for tumor progression. Based on qualitative in situ hybridization studies in human cancer tissue, a range of components involved in proteolysis appear to be expressed by stromal cells rather than cancer...

  11. The coagulation factor Xa/protease activated receptor-2 axis in the progression of liver fibrosis : a multifaceted paradigm

    NARCIS (Netherlands)

    Borensztajn, Keren; von der Thusen, Jan H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

    2010-01-01

    Introduction Activation of the coagulation cascade during liver fibrosis: a puzzling paradox Protease-activated receptors: the link between coagulation cascade activation and liver fibrosis Expression and distribution of human PAR-2 in normal and pathological liver tissue FXa signalling on PAR-2

  12. Mechanisms and cellular functions of intramembrane proteases.

    Science.gov (United States)

    Urban, Siniša

    2013-12-01

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

  13. Changes in IgA protease expression are conferred by changes in genomes during persistent infection by nontypeable Haemophilus influenzae in chronic obstructive pulmonary disease.

    Science.gov (United States)

    Gallo, Mary C; Kirkham, Charmaine; Eng, Samantha; Bebawee, Remon S; Kong, Yong; Pettigrew, Melinda M; Tettelin, Hervé; Murphy, Timothy F

    2018-05-14

    Nontypeable Haemophilus influenzae (NTHi) is an exclusively human pathobiont that plays a critical role in the course and pathogenesis of chronic obstructive pulmonary disease (COPD). NTHi causes acute exacerbations of COPD and also causes persistent infection of the lower airways. NTHi expresses four IgA protease variants (A1, A2, B1, and B2) that play different roles in virulence. Expression of IgA proteases varies among NTHi strains, but little is known about the frequency and mechanisms by which NTHi modulates IgA protease expression during infection in COPD. To assess expression of IgA protease during natural infection in COPD, we studied IgA protease expression of 101 persistent strains (median duration of persistence 161 days, range 2 to 1422) collected longitudinally from patients enrolled in a 20-year study of COPD upon initial acquisition and immediately before clearance from the host. Upon acquisition, 89 (88%) expressed IgA protease. A total of 16 of 101 (16%) strains of NTHi altered expression of IgA protease during persistence. Indels and slipped-strand mispairing of mononucleotide repeats conferred changes in expression of igaA1, igaA2, and igaB1 Strains with igaB2 underwent frequent changes in expression of IgA protease B2 during persistence, mediated by slipped-strand mispairing of a 7-nucleotide repeat, TCAAAAT, within the open reading frame of igaB2 We conclude that changes in iga gene sequences result in changes in expression of IgA proteases by NTHi during persistent infection in the respiratory tract of patients with COPD. Copyright © 2018 American Society for Microbiology.

  14. Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

    Science.gov (United States)

    Chatel-Chaix, Laurent; Baril, Martin; Lamarre, Daniel

    2010-01-01

    Hepatitis C virus (HCV) infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease) that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection. PMID:21994705

  15. Hepatitis C Virus NS3/4A Protease Inhibitors: A Light at the End of the Tunnel

    Directory of Open Access Journals (Sweden)

    Laurent Chatel-Chaix

    2010-08-01

    Full Text Available Hepatitis C virus (HCV infection is a serious and growing threat to human health. The current treatment provides limited efficacy and is poorly tolerated, highlighting the urgent medical need for novel therapeutics. The membrane-targeted NS3 protein in complex with the NS4A comprises a serine protease domain (NS3/4A protease that is essential for viral polyprotein maturation and contributes to the evasion of the host innate antiviral immunity by HCV. Therefore, the NS3/4A protease represents an attractive target for drug discovery, which is tied in with the challenge to develop selective small-molecule inhibitors. A rational drug design approach, based on the discovery of N-terminus product inhibition, led to the identification of potent and orally bioavailable NS3 inhibitors that target the highly conserved protease active site. This review summarizes the NS3 protease inhibitors currently challenged in clinical trials as one of the most promising antiviral drug class, and possibly among the first anti-HCV agents to be approved for the treatment of HCV infection.

  16. ReFlexIn: a flexible receptor protein-ligand docking scheme evaluated on HIV-1 protease.

    Directory of Open Access Journals (Sweden)

    Simon Leis

    Full Text Available For many targets of pharmaceutical importance conformational changes of the receptor protein are relevant during the ligand binding process. A new docking approach, ReFlexIn (Receptor Flexibility by Interpolation, that combines receptor flexibility with the computationally efficient potential grid representation of receptor molecules has been evaluated on the retroviral HIV-1 (Human Immunodeficiency Virus 1 protease system. An approximate inclusion of receptor flexibility is achieved by using interpolation between grid representations of individual receptor conformations. For the retroviral protease the method was tested on an ensemble of protease structures crystallized in the presence of different ligands and on a set of structures obtained from morphing between the unbound and a ligand-bound protease structure. Docking was performed on ligands known to bind to the protease and several non-binders. For the binders the ReFlexIn method yielded in almost all cases ligand placements in similar or closer agreement with experiment than docking to any of the ensemble members without degrading the discrimination with respect to non-binders. The improved docking performance compared to docking to rigid receptors allows for systematic virtual screening applications at very small additional computational cost.

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

    African Journals Online (AJOL)

    Nasir

    2016-10-12

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

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

    NARCIS (Netherlands)

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

    2017-01-01

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

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

    African Journals Online (AJOL)

    STORAGESEVER

    2008-05-16

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

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

    African Journals Online (AJOL)

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

  1. Isolation of alkaline protease from Bacillus subtilis AKRS3

    African Journals Online (AJOL)

    ashok

    2012-08-28

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

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

    NARCIS (Netherlands)

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

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

  3. Cold denaturation of the HIV-1 protease monomer

    DEFF Research Database (Denmark)

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

    2017-01-01

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

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

    AJL

    2012-02-16

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

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

    African Journals Online (AJOL)

    user

    2011-02-14

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

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

    African Journals Online (AJOL)

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

  11. Crimean-Congo Hemorrhagic Fever Virus Suppresses Innate Immune Responses via a Ubiquitin and ISG15 Specific Protease

    Directory of Open Access Journals (Sweden)

    Florine E.M. Scholte

    2017-09-01

    Full Text Available Antiviral responses are regulated by conjugation of ubiquitin (Ub and interferon-stimulated gene 15 (ISG15 to proteins. Certain classes of viruses encode Ub- or ISG15-specific proteases belonging to the ovarian tumor (OTU superfamily. Their activity is thought to suppress cellular immune responses, but studies demonstrating the function of viral OTU proteases during infection are lacking. Crimean-Congo hemorrhagic fever virus (CCHFV, family Nairoviridae is a highly pathogenic human virus that encodes an OTU with both deubiquitinase and deISGylase activity as part of the viral RNA polymerase. We investigated CCHFV OTU function by inactivating protease catalytic activity or by selectively disrupting its deubiquitinase and deISGylase activity using reverse genetics. CCHFV OTU inactivation blocked viral replication independently of its RNA polymerase activity, while deubiquitinase activity proved critical for suppressing the interferon responses. Our findings provide insights into viral OTU functions and support the development of therapeutics and vaccines.

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

    Science.gov (United States)

    Panigrahi, Rashmi; Lemieux, M Joanne

    2015-01-01

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

  13. Improved Protease-Targeting and Biopharmaceutical Properties of Novel Prodrugs of Ganciclovir.

    Science.gov (United States)

    Sun, Kefeng; Xu, Hao; Hilfinger, John L; Lee, Kyung-Dall; Provoda, Chester J; Sabit, Hairat; Amidon, Gordon L

    2018-02-05

    The prodrug strategy has been frequently employed as a chemical approach for overcoming the disadvantages of existing parent drugs. In this report, we synthesized four monoester prodrugs of ganciclovir, an anticytomegalovirus drug, and demonstrated their potential advantages in protease-targeted activation and biopharmaceutical profiles over the parent compound. We demonstrated that these four prodrugs of ganciclovir, i.e., N-benzyloxycarbonyl-(L)-alanine-ganciclovir (CbzAlaGCV), N-benzyloxycarbonyl-(α,l)-aminobutyric acid-ganciclovir (CbzAbuGCV), N-acetyl-(l)-phenylalanine-(l)-alanine-ganciclovir (AcPheAlaGCV), and N-acetyl-(l)-phenylalanine-(α,l)-aminobutyric acid-ganciclovir (AcPheAbuGCV), are hydrolytically activated by the protease of human cytomegalovirus (hCMV), a serine protease that possesses intrinsic esterase activities. CbzAlaGCV and AcPheAlaGCV were found to be activated at a higher rate by the hCMV protease than CbzAbuGCV and AcPheAbuGCV. These ganciclovir prodrugs could potentially be targeted to selective activation by the hCMV protease which is only present at the viral infection sites, thereby achieving higher efficacy and lower systemic toxicity. The tissue stability, cellular uptake, and trans-epithelial transport of these ganciclovir prodrugs were also characterized. The N-acetylated dipeptide prodrugs of ganciclovir were found to be generally more stable than Cbz-amino acid prodrugs in various tissue matrices. Among the four prodrug candidates, AcPheAbuGCV was the most stable in human cell homogenates, plasma, and pooled liver microsomes. AcPheAbuGCV also possessed a superior cellular uptake profile and permeability across epithelial cell monolayers. Since the targeting and selective activation of a prodrug is determined by not only its rate of hydrolysis catalyzed by the hCMV protease target but also its biopharmaceutical properties, i.e., oral absorption and systemic availability, AcPheAbuGCV is considered the best overall candidate among

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

    Directory of Open Access Journals (Sweden)

    Sowdhamini R

    2006-08-01

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

  15. Molecular cloning and tissue-specific expression analysis of mouse spinesin, a type II transmembrane serine protease 5

    International Nuclear Information System (INIS)

    Watanabe, Yoshihisa; Okui, Akira; Mitsui, Shinichi; Kawarabuki, Kentaro; Yamaguchi, Tatsuyuki; Uemura, Hidetoshi; Yamaguchi, Nozomi

    2004-01-01

    We have previously reported novel serine proteases isolated from cDNA libraries of the human and mouse central nervous system (CNS) by PCR using degenerate oligodeoxyribonucleotide primers designed on the basis of the serine protease motifs, AAHC and DSGGP. Here we report a newly isolated serine protease from the mouse CNS. This protease is homologous (77.9% identical) to human spinesin type II transmembrane serine protease 5. Mouse spinesin (m-spinesin) is also composed of (from the N-terminus) a short cytoplasmic domain, a transmembrane domain, a stem region containing a scavenger-receptor-like domain, and a serine protease domain, as is h-spinesin. We also isolated type 1, type 2, and type 3 variant cDNAs of m-spinesin. Full-length spinesin (type 4) and type 3 contain all the domains, whereas type 1 and type 2 variants lack the cytoplasmic, transmembrane, and scavenger-receptor-like domains. Subcellular localization of the variant forms was analyzed using enhanced green fluorescent protein (EGFP) fusion proteins. EGFP-type 4 fusion protein was predominantly localized to the ER, Golgi apparatus, and plasma membrane, whereas EGFP-type 1 was localized to the cytoplasm, reflecting differential classification of m-spinesin variants into transmembrane and cytoplasmic types. We analyzed the distribution of m-spinesin variants in mouse tissues, using RT-PCR with variant-specific primer sets. Interestingly, transmembrane-type spinesin, types 3 and 4, was specifically expressed in the spinal cord, whereas cytoplasmic type, type 1, was expressed in multiple tissues, including the cerebrum and cerebellum. Therefore, m-spinesin variants may have distinct biological functions arising from organ-specific variant expression

  16. A family of serine proteases of Clavibacter michiganensis subsp. michiganensis: chpC plays a role in colonization of the host plant tomato.

    Science.gov (United States)

    Stork, Ines; Gartemann, Karl-Heinz; Burger, Annette; Eichenlaub, Rudolf

    2008-09-01

    Genes for seven putative serine proteases (ChpA-ChpG) belonging to the trypsin subfamily and homologous to the virulence factor pat-1 were identified on the chromosome of Clavibacter michiganensis subsp. michiganensis (Cmm) NCPPB382. All proteases have signal peptides indicating export of these proteins. Their putative function is suggested by two motifs and an aspartate residue typical for serine proteases. Furthermore, six cysteine residues are located at conserved positions. The genes are clustered in a chromosomal region of about 50 kb with a significantly lower G + C content than common for Cmm. The genes chpA, chpB and chpD are pseudogenes as they contain frame shifts and/or in-frame stop codons. The genes chpC and chpG were inactivated by the insertion of an antibiotic resistance cassette. The chpG mutant was not impaired in virulence. However, in planta the titre of the chpC mutant was drastically reduced and only weak disease symptoms were observed. Complementation of the chpC mutant by the wild-type allele restored full virulence. ChpC is the first chromosomal gene of Cmm identified so far that affects the interaction of the pathogen with the host plant.

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

    Science.gov (United States)

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

    2007-03-09

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

  18. Salmon trypsin stimulates the expression of interleukin-8 via protease-activated receptor-2

    International Nuclear Information System (INIS)

    Larsen, Anett K.; Seternes, Ole-Morten; Larsen, Merethe; Aasmoe, Lisbeth; Bang, Berit

    2008-01-01

    In this study, we focus on salmon trypsin as an activator of inflammatory responses in airway cells in vitro. The rationale behind the investigation is that salmon industry workers are exposed to aerosols containing enzymes, which are generated during industrial processing of the fish. Knowing that serine proteases such as trypsin are highly active mediators with diverse biological activities, the stimulation of nuclear factor-kappa B (NF-κB) and interleukin (IL)-8 and the role of protease-activated receptors (PAR) in inflammatory signal mediation were investigated. Protease-activated receptors are considered important under pathological situations in the human airways, and a thorough understanding of PAR-induced cellular events and their consequences in airway inflammation is necessary. Human airway epithelial cells (A549) were exposed to trypsin isolated from fish (Salmo salar), and we observed that purified salmon trypsin could generate secretion of IL-8 in a concentration-dependent manner. Furthermore, we demonstrate that PAR-2 activation by salmon trypsin is coupled to an induction of NF-κB-mediated transcription using a PAR-2 transfected HeLa cell model. Finally, we show that the release of IL-8 from A549 following stimulation with purified salmon trypsin is mediated through activation of PAR-2 using specific small interfering RNAs (siRNAs). The results presented suggest that salmon trypsin, via activation of PAR-2, might influence inflammation processes in the airways if inhaled in sufficient amounts

  19. The effect of postirradiation application of aspartic acid salts on hemopoietic recovery in sublethally X-irradiated mice

    International Nuclear Information System (INIS)

    Pospisil, M.; Netikova, J.; Vasku, J.; Urbanek, E.

    1979-01-01

    The effect of aspartic acid salts, especially of K and Mg aspartates, on certain hematological changes in the peripheral blood and hemopoietic organs of sublethally X-irratiated male mice of the strain C57Bl/10 was investigated. Salts of aspartic acid were administered in tap water after irradiation. A favorable effect of aspartic acid salts on erythropoietic recovery and on regeneration of thymus weight was found during the first two weeks after irradiation. (orig.) [de

  20. Finding a Leucine in a Haystack: Searching the Proteome for ambigous Leucine-Aspartic Acid motifs

    KAUST Repository

    Arold, Stefan T.

    2016-01-25

    Leucine-aspartic acid (LD) motifs are short helical protein-protein interaction motifs involved in cell motility, survival and communication. LD motif interactions are also implicated in cancer metastasis and are targeted by several viruses. LD motifs are notoriously difficult to detect because sequence pattern searches lead to an excessively high number of false positives. Hence, despite 20 years of research, only six LD motif–containing proteins are known in humans, three of which are close homologues of the paxillin family. To enable the proteome-wide discovery of LD motifs, we developed LD Motif Finder (LDMF), a web tool based on machine learning that combines sequence information with structural predictions to detect LD motifs with high accuracy. LDMF predicted 13 new LD motifs in humans. Using biophysical assays, we experimentally confirmed in vitro interactions for four novel LD motif proteins. Thus, LDMF allows proteome-wide discovery of LD motifs, despite a highly ambiguous sequence pattern. Functional implications will be discussed.

  1. Multiphoton manipulations of enzymatic photoactivity in aspartate aminotransferase.

    Science.gov (United States)

    Hill, Melissa P; Freer, Lucy H; Vang, Mai C; Carroll, Elizabeth C; Larsen, Delmar S

    2011-04-21

    The aspartate aminotransferase (AAT) enzyme utilizes the chromophoric pyridoxal 5'-phosphate (PLP) cofactor to facilitate the transamination of amino acids. Recently, we demonstrated that, upon exposure to blue light, PLP forms a reactive triplet state that rapidly (in microseconds) generates the high-energy quinonoid intermediate when bound to PLP-dependent enzymes [J. Am. Chem. Soc.2010, 132 (47), 16953-16961]. This increases the net catalytic activity (k(cat)) of AAT, since formation of the quinonoid is partially rate limiting via the thermally activated enzymatic pathway. The magnitude of observed photoenhancement initially scales linearly with pump fluence; however when a critical threshold is exceeded, the photoactivity saturates and is even suppressed at greater excitation fluences. The photodynamic mechanisms associated with this suppression behavior are characterized with the use of ultrafast multipulse pump-dump-probe and pump-repump-probe transient absorption techniques in combination with complementary two-color, steady-state excitation assays. Via multistate kinetic modeling of the transient ultrafast data and the steady-state assay data, the nonmonotonic incident power dependence of the photoactivty in AAT is decomposed into contributions from high-intensity dumping of the excited singlet state and repumping of the excited triplet state with induces the repopulation of the ground state via rapid intersystem crossing in the higher-lying triplet electronic manifold.

  2. Supermacroporous chemically cross-linked poly(aspartic acid) hydrogels.

    Science.gov (United States)

    Gyarmati, Benjámin; Mészár, E Zsuzsanna; Kiss, Lóránd; Deli, Mária A; László, Krisztina; Szilágyi, András

    2015-08-01

    Chemically cross-linked poly(aspartic acid) (PASP) gels were prepared by a solid-liquid phase separation technique, cryogelation, to achieve a supermacroporous interconnected pore structure. The precursor polymer of PASP, polysuccinimide (PSI) was cross-linked below the freezing point of the solvent and the forming crystals acted as templates for the pores. Dimethyl sulfoxide was chosen as solvent instead of the more commonly used water. Thus larger temperatures could be utilized for the preparation and the drawback of increase in specific volume of water upon freezing could be eliminated. The morphology of the hydrogels was characterized by scanning electron microscopy and interconnectivity of the pores was proven by the small flow resistance of the gels. Compression tests also confirmed the interconnected porous structure and the complete re-swelling and shape recovery of the supermacroporous PASP hydrogels. The prepared hydrogels are of interest for several biomedical applications as scaffolding materials because of their cytocompatibility, controllable morphology and pH-responsive character. Copyright © 2015 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  3. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Casablanca cohort of the A 1 chieve study

    Directory of Open Access Journals (Sweden)

    Ahmed Farouqi

    2013-01-01

    Full Text Available Background: The A 1 chieve, a multicentric (28 countries, 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726 in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Casablanca, Morocco. Results: A total of 495 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Study patients had started on or were switched to biphasic insulin aspart (n = 231, insulin detemir (n = 151, insulin aspart (n = 19, basal insulin plus insulin aspart (n = 53 and other insulin combinations (n = 41. At baseline glycaemic control was poor for both insulin naïve (mean HbA 1 c: 10.2% and insulin user (mean HbA 1 c: 9.4% groups. After 24 weeks of treatment, both groups showed improvement in HbA 1 c (insulin naïve: −2.3%, insulin users: −1.8%. Major hypoglycaemia was observed in the insulin naïve group after 24 weeks. SADRs were reported in 1.2% of insulin naïve and 2.1% of insulin user groups. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia.

  4. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Kolkata cohort of the A 1 chieve study

    Directory of Open Access Journals (Sweden)

    Anirban Majumder

    2013-01-01

    Full Text Available Background: The A 1 chieve, a multicentric (28 countries, 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726 in routine clinical care across four continents. Materials and Methods: Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Kolkata, India. Results: A total of 576 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Patients had started on or were switched to biphasic insulin aspart (n = 417, insulin detemir (n = 70, insulin aspart (n = 55, basal insulin plus insulin aspart (n = 19 and other insulin combinations (n = 15. At baseline, glycaemic control was poor for both insulin naïve (mean HbA 1 c: 8.3% and insulin user (mean HbA 1 c: 8.6% groups. After 24 weeks of treatment, both the groups showed improvement in HbA 1 c (insulin naïve: −1.3%, insulin users: −1.4%. SADRs including major hypoglycaemic events or episodes did not occur in any of the study patients. Conclusion: Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia.

  5. Clinical experience with insulin detemir, biphasic insulin aspart and insulin aspart in people with type 2 diabetes: Results from the Mumbai cohort of the A1chieve study.

    Science.gov (United States)

    Talwalkar, P G; Gupta, Vishal; Kovil, Rajiv

    2013-11-01

    The A1chieve, a multicentric (28 countries), 24-week, non-interventional study evaluated the safety and effectiveness of insulin detemir, biphasic insulin aspart and insulin aspart in people with T2DM (n = 66,726) in routine clinical care across four continents. Data was collected at baseline, at 12 weeks and at 24 weeks. This short communication presents the results for patients enrolled from Mumbai, India. A total of 2112 patients were enrolled in the study. Four different insulin analogue regimens were used in the study. Patients had started on or were switched to biphasic insulin aspart (n = 1561), insulin detemir (n = 313), insulin aspart (n = 144), basal insulin plus insulin aspart (n = 53) and other insulin combinations (n = 41). At baseline glycaemic control was poor for both insulin naïve (mean HbA1c: 8.7%) and insulin user (mean HbA1c: 9.2%) groups. After 24 weeks of treatment, both the groups showed improvement in HbA1c (insulin naïve: -1.4%, insulin users: -1.8%). SADRs including major hypoglycaemic events or episodes did not occur in any of the study patients. Starting or switching to insulin analogues was associated with improvement in glycaemic control with a low rate of hypoglycaemia.

  6. Highly potent fibrinolytic serine protease from Streptomyces.

    Science.gov (United States)

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

    2011-01-05

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

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

    Science.gov (United States)

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

    1999-09-01

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

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

    Directory of Open Access Journals (Sweden)

    E. V. Маtseliukh

    2015-04-01

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

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

    Science.gov (United States)

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

    2017-01-01

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

  10. Hyper production of alkaline protease by mutagenized bacillus subtilis

    International Nuclear Information System (INIS)

    Qureshi, A.M.; Tanseem, F.

    2010-01-01

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

  11. Optimizing PHB and Protease Production by Box Behnken Design

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    Amro Abd al fattah Amara

    2013-04-01

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

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

    Directory of Open Access Journals (Sweden)

    Lilburn Timothy G

    2011-12-01

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

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

    Science.gov (United States)

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

    2016-06-01

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

  14. Giardia duodenalis Surface Cysteine Proteases Induce Cleavage of the Intestinal Epithelial Cytoskeletal Protein Villin via Myosin Light Chain Kinase.

    Directory of Open Access Journals (Sweden)

    Amol Bhargava

    Full Text Available Giardia duodenalis infections are among the most common causes of waterborne diarrhoeal disease worldwide. At the height of infection, G. duodenalis trophozoites induce multiple pathophysiological processes within intestinal epithelial cells that contribute to the development of diarrhoeal disease. To date, our understanding of pathophysiological processes in giardiasis remains incompletely understood. The present study reveals a previously unappreciated role for G. duodenalis cathepsin cysteine proteases in intestinal epithelial pathophysiological processes that occur during giardiasis. Experiments first established that Giardia trophozoites indeed produce cathepsin B and L in strain-dependent fashion. Co-incubation of G. duodenalis with human enterocytes enhanced cathepsin production by Assemblage A (NF and S2 isolates trophozoites, but not when epithelial cells were exposed to Assemblage B (GSM isolate trophozoites. Direct contact between G. duodenalis parasites and human intestinal epithelial monolayers resulted in the degradation and redistribution of the intestinal epithelial cytoskeletal protein villin; these effects were abolished when parasite cathepsin cysteine proteases were inhibited. Interestingly, inhibition of parasite proteases did not prevent degradation of the intestinal tight junction-associated protein zonula occludens 1 (ZO-1, suggesting that G. duodenalis induces multiple pathophysiological processes within intestinal epithelial cells. Finally, this study demonstrates that G. duodenalis-mediated disruption of villin is, at least, in part dependent on activation of myosin light chain kinase (MLCK. Taken together, this study indicates a novel role for parasite cathepsin cysteine proteases in the pathophysiology of G. duodenalis infections.

  15. L-aspartic acid transport by cat erythrocytes

    International Nuclear Information System (INIS)

    Chen, C.W.; Preston, R.L.

    1986-01-01

    Cat and dog red cells are unusual in that they have no Na/K ATPase and contain low K and high Na intracellularly. They also show significant Na dependent L-aspartate (L-asp) transport. The authors have characterized this system in cat RBCs. The influx of 3 H-L-asp (typically 2μM) was measured in washed RBCs incubated for 60 s at 37 0 C in medium containing 140 mM NaCl, 5 mM Kcl, 2 mM CaCl 2 , 15 mM MOPS pH 7.4, 5 mM glucose, and 14 C-PEG as a space marker. The cells were washed 3 times in the medium immediately before incubation which was terminated by centrifuging the RBCs through a layer of dibutylphthalate. Over an L-asp concentration range of 0.5-1000μM, influx obeyed Michaelis-Menten kinetics with a small added linear diffusion component. The Kt and Jmax of the saturable component were 5.40 +/- 0.34 μM and 148.8 +/- 7.2 μmol 1. cell -1 h -1 respectively. Replacement of Na with Li, K, Rb, Cs or choline reduce influx to diffusion. With the addition of asp analogues (4 + M L-asp, 40 + M inhibitor), the following sequence of inhibition was observed (range 80% to 40% inhib.): L-glutamate > L-cysteine sulfonate > D-asp > L-cysteic acid > D-glutamate. Other amino acids such as L-alanine, L-proline, L-lysine, L-cysteine, and taurine showed no inhibition (<5%). These data suggest that cat red cells contain a high-affinity Na dependent transport system for L-asp, glutamate, and closely related analogues which resembles that found in the RBCs of other carnivores and in neural tissues

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

    Science.gov (United States)

    Sharma, Ranu; Suresh, C G

    2015-01-01

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

  17. Preparation and functional evaluation of collagen oligopeptide-rich hydrolysate from fish skin with the serine collagenolytic protease from Pseudoalteromonas sp. SM9913.

    Science.gov (United States)

    Chen, Xiu-Lan; Peng, Ming; Li, Jing; Tang, Bai-Lu; Shao, Xuan; Zhao, Fang; Liu, Chang; Zhang, Xi-Ying; Li, Ping-Yi; Shi, Mei; Zhang, Yu-Zhong; Song, Xiao-Yan

    2017-11-16

    Although several serine collagenolytic proteases from bacteria were reported, none has been used to prepare bioactive collagen peptides. MCP-01 is the most abundant extracellular protease of deep-sea Pseudoalteromonas sp. SM9913 and is a serine collagenolytic protease with high efficiency on fish collagen hydrolysis. Here, we set up a pilot scale process to ferment SM9913 for extracellular protease production. With SM9913 extracellular protease as a tool, a process to prepare collagen oligopeptide-rich hydrolysate from codfish skin was set up, which was further scaled up to pilot (100 L) and plant (2000 L) levels with yields >66%. The hydrolysates from laboratory-, pilot- and plant-scales had quite similar quality, containing ~95% peptides with molecular weights lower than 3000 Da and approximately 60% lower than 1000 Da, in which collagen oilgopeptides account for approximately 95%. Bioactivity analyses showed that the hydrolysate had moisture-retention ability, antioxidant activity, and promoting effect on cell viability of human dermal fibroblasts. Safety evaluation showed that the hydrolysate was nontoxic and nonirritating to skin. Therefore, SM9913 extracellular protease is a good enzyme to prepare bioactive oligopeptides from fish skin. The results also suggest that the collagen oligopeptides-rich hydrolysate may have potentials in biomedical, functional food, pharmaceutical and cosmetic industries.

  18. 78 FR 67365 - Determination That Adderall (Amphetamine Aspartate; Amphetamine Sulfate; Dextroamphetamine...

    Science.gov (United States)

    2013-11-12

    ... the Drug Price Competition and Patent Term Restoration Act of 1984 (Pub. L. 98-417) (the 1984... No. Drug Applicant NDA 011522 ADDERALL Teva Womens Health (amphetamine Inc., 41 Moores aspartate; Rd...

  19. Supporting Aspartate Biosynthesis Is an Essential Function of Respiration in Proliferating Cells.

    Science.gov (United States)

    Sullivan, Lucas B; Gui, Dan Y; Hosios, Aaron M; Bush, Lauren N; Freinkman, Elizaveta; Vander Heiden, Matthew G

    2015-07-30

    Mitochondrial respiration is important for cell proliferation; however, the specific metabolic requirements fulfilled by respiration to support proliferation have not been defined. Here, we show that a major role of respiration in proliferating cells is to provide electron acceptors for aspartate synthesis. This finding is consistent with the observation that cells lacking a functional respiratory chain are auxotrophic for pyruvate, which serves as an exogenous electron acceptor. Further, the pyruvate requirement can be fulfilled with an alternative electron acceptor, alpha-ketobutyrate, which provides cells neither carbon nor ATP. Alpha-ketobutyrate restores proliferation when respiration is inhibited, suggesting that an alternative electron acceptor can substitute for respiration to support proliferation. We find that electron acceptors are limiting for producing aspartate, and supplying aspartate enables proliferation of respiration deficient cells in the absence of exogenous electron acceptors. Together, these data argue a major function of respiration in proliferating cells is to support aspartate synthesis. Copyright © 2015 Elsevier Inc. All rights reserved.

  20. Improved tolerance of abdominal large-volume radiotherapy due to ornithine aspartate

    International Nuclear Information System (INIS)

    Kuttig, H.

    1983-01-01

    The influence of ornithine aspartate on supporting the hepatic function was investigated in a group of 47 patients with tumour dissemination in the pelvic and abdominal region, randomised on the basis of the progress of the serum enzymes GOT, GPT, LAD, LDH, LAP and the alkaline phosphatase during and following completion of a course of large-volume radiotherapy. The adjuvant therapy with ornithine aspartate resulted in reduced enzyme movement with an earlier tendency to normalisation. The results, which are borne out by statistics, clearly show an improvement in the hepatic function on detoxication of toxic degradation products of radiotherapy with reduced impairment of the body's own defence mechanisms. Subjectively too, the course of treatment with ornithine aspartate showed a reduced ratio of side effects as regards lassitude and impairment of the patient's general well-being as compared with the group of patients to whom ornithine aspartate was not simultaneously administered. (orig.) [de

  1. Chiral Asymmetric Structures in Aspartic Acid and Valine Crystals Assessed by Atomic Force Microscopy.

    Science.gov (United States)

    Teschke, Omar; Soares, David Mendez

    2016-03-29

    Structures of crystallized deposits formed by the molecular self-assembly of aspartic acid and valine on silicon substrates were imaged by atomic force microscopy. Images of d- and l-aspartic acid crystal surfaces showing extended molecularly flat sheets or regions separated by single molecule thick steps are presented. Distinct orientation surfaces were imaged, which, combined with the single molecule step size, defines the geometry of the crystal. However, single molecule step growth also reveals the crystal chirality, i.e., growth orientations. The imaged ordered lattice of aspartic acid (asp) and valine (val) mostly revealed periodicities corresponding to bulk terminations, but a previously unreported molecular hexagonal lattice configuration was observed for both l-asp and l-val but not for d-asp or d-val. Atomic force microscopy can then be used to identify the different chiral forms of aspartic acid and valine crystals.

  2. msaABCR operon positively regulates biofilm development by repressing proteases and autolysis in Staphylococcus aureus.

    Science.gov (United States)

    Sahukhal, Gyan S; Batte, Justin L; Elasri, Mohamed O

    2015-02-01

    Staphylococcus aureus is an important human pathogen that causes nosocomial and community-acquired infections. One of the most important aspects of staphylococcal infections is biofilm development within the host, which renders the bacterium resistant to the host's immune response and antimicrobial agents. Biofilm development is very complex and involves several regulators that ensure cell survival on surfaces within the extracellular polymeric matrix. Previously, we identified the msaABCR operon as an additional positive regulator of biofilm formation. In this study, we define the regulatory pathway by which msaABCR controls biofilm formation. We demonstrate that the msaABCR operon is a negative regulator of proteases. The control of protease production mediates the processing of the major autolysin, Atl, and thus regulates the rate of autolysis. In the absence of the msaABCR operon, Atl is processed by proteases at a high rate, leading to increased cell death and a defect in biofilm maturation. We conclude that the msaABCR operon plays a key role in maintaining the balance between autolysis and growth within the staphylococcal biofilm. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Suppressing IL-36-driven inflammation using peptide pseudosubstrates for neutrophil proteases.

    Science.gov (United States)

    Sullivan, Graeme P; Henry, Conor M; Clancy, Danielle M; Mametnabiev, Tazhir; Belotcerkovskaya, Ekaterina; Davidovich, Pavel; Sura-Trueba, Sylvia; Garabadzhiu, Alexander V; Martin, Seamus J

    2018-03-07

    Sterile inflammation is initiated by molecules released from necrotic cells, called damage-associated molecular patterns (DAMPs). Members of the extended IL-1 cytokine family are important DAMPs, are typically only released through necrosis, and require limited proteolytic processing for activation. The IL-1 family cytokines, IL-36α, IL-36β, and IL-36γ, are expressed as inactive precursors and have been implicated as key initiators of psoriatic-type skin inflammation. We have recently found that IL-36 family cytokines are proteolytically processed and activated by the neutrophil granule-derived proteases, elastase, and cathepsin G. Inhibitors of IL-36 processing may therefore have utility as anti-inflammatory agents through suppressing activation of the latter cytokines. We have identified peptide-based pseudosubstrates for cathepsin G and elastase, based on optimal substrate cleavage motifs, which can antagonize activation of all three IL-36 family cytokines by the latter proteases. Human psoriatic skin plaques displayed elevated IL-36β processing activity that could be antagonized by peptide pseudosubstrates specific for cathepsin G. Thus, antagonists of neutrophil-derived proteases may have therapeutic potential for blocking activation of IL-36 family cytokines in inflammatory conditions such as psoriasis.

  4. Negative regulation of quorum-sensing systems in Pseudomonas aeruginosa by ATP-dependent Lon protease.

    Science.gov (United States)

    Takaya, Akiko; Tabuchi, Fumiaki; Tsuchiya, Hiroko; Isogai, Emiko; Yamamoto, Tomoko

    2008-06-01

    Lon protease, a member of the ATP-dependent protease family, regulates numerous cellular systems by degrading specific substrates. Here, we demonstrate that Lon is involved in the regulation of quorum-sensing (QS) signaling systems in Pseudomonas aeruginosa, an opportunistic human pathogen. The organism has two acyl-homoserine lactone (HSL)-mediated QS systems, LasR/LasI and RhlR/RhlI. Many reports have demonstrated that these two systems are regulated and interconnected by global regulators. We found that lon-disrupted cells overproduce pyocyanin, the biosynthesis of which depends on the RhlR/RhlI system, and show increased levels of a transcriptional regulator, RhlR. The QS systems are organized hierarchically: the RhlR/RhlI system is subordinate to LasR/LasI. To elucidate the mechanism by which Lon negatively regulates RhlR/RhlI, we examined the effect of lon disruption on the LasR/LasI system. We found that Lon represses the expression of LasR/LasI by degrading LasI, an HSL synthase, leading to negative regulation of the RhlR/RhlI system. RhlR/RhlI was also shown to be regulated by Lon independently of LasR/LasI via regulation of RhlI, an HSL synthase. In view of these findings, it is suggested that Lon protease is a powerful negative regulator of both HSL-mediated QS systems in P. aeruginosa.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2008-10-27

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

  6. Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

    Directory of Open Access Journals (Sweden)

    Thomas A Prohaska

    Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

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

    OpenAIRE

    Naiola, Elidar; Widhyastuti, Nunuk

    2002-01-01

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

  8. N-acetyl Aspartate Levels in Adolescents With Bipolar and/or Cannabis Use Disorders

    Science.gov (United States)

    Bitter, Samantha M.; Weber, Wade A.; Chu, Wen-Jang; Adler, Caleb M.; Eliassen, James C.; Strakowski, Stephen M.; DelBello, Melissa P.

    2014-01-01

    Objective Bipolar and cannabis use disorders commonly co-occur during adolescence, and neurochemical studies may help clarify the pathophysiology underlying this co-occurrence. This study compared metabolite concentrations in the left ventral lateral prefrontal cortex among: adolescents with bipolar disorder (bipolar group; n=14), adolescents with a cannabis use disorder (cannabis use group, n=13), adolescents with cannabis use and bipolar disorders (bipolar and cannabis group, n=25), and healthy adolescents (healthy controls, n=15). We hypothesized that adolescents with bipolar disorder (with or without cannabis use disorder) would have decreased N-acetyl aspartate levels in the ventral lateral prefrontal cortex compared to the other groups, and that the bipolar and cannabis group would have the lowest N-acetyl aspartate levels of all groups. Methods N-acetyl aspartate concentrations in the left ventral lateral prefrontal cortex were obtained using Proton Magnetic Resonance Spectroscopy. Results Adolescents with bipolar disorder showed significantly lower left ventral lateral prefrontal cortex N-acetyl aspartate levels, but post-hoc analyses indicated that this was primarily due to increased N-acetyl aspartate levels in the cannabis group. The cannabis use disorder group had significantly higher N-acetyl aspartate levels compared to the bipolar disorder and the bipolar and cannabis groups (p=0.0002 and p=0.0002, respectively). Pearson correlations revealed a significant positive correlation between amount of cannabis used and N-acetyl aspartate concentrations. Conclusions Adolescents with cannabis use disorder showed higher levels of N-acetyl aspartate concentrations that were significantly positively associated with the amount of cannabis used; however, this finding was not present in adolescents with comorbid bipolar disorder. PMID:24729763

  9. NMR and spectroscopic studies on uranyl ion interaction with aspartic acid and asparagine

    International Nuclear Information System (INIS)

    Wieczorek, H.; Kozlowski, H.

    1980-01-01

    The carboxyl groups of peptides or proteins are quite effective in the binding of UO 2 +2 ion and as the first step in studies in that field aspartic acid has been chosen as the bi-carboxylic ligand. The data for UO 2 +2 -asparagine system are also presented in this communication as they complete the results obtained for the UO 2 +2 -aspartic acid system. (author)

  10. A cluster of aspartic residues in the extracellular loop II of PAR 4 is important for thrombin interaction and activation of platelets.

    Science.gov (United States)

    Sánchez Centellas, Daniel; Gudlur, Sushanth; Vicente-Carrillo, Alejandro; Ramström, Sofia; Lindahl, Tomas L

    2017-06-01

    Thrombin activates platelets via proteolytic cleavage of protease-activated receptors (PARs) 1 and 4. The two PARs have distinct but complementary roles. The mechanisms responsible for PAR1 activation by thrombin have been extensively studied. However, much less is known regarding thrombin activation of PAR4, especially the potential involvement of regions of PAR4 other than the N-terminal, which is bound to the catalytic site of thrombin. We have studied PAR4 in S. cerevisiae strain MMY12, an expression system in which the GPCR receptors are connected to a Lac Z reporter gene resulting in increased β-galactosidase activity. This approach was used to assess PAR4 mutants to evaluate the contribution of different aspartic residues in facilitating PAR4 activation. Furthermore, peptides mimicking parts of the PAR4 N-terminal and the second extracellular loop (ECLII) were tested for their ability to inhibit platelet activation by thrombin. Binding of these peptides to γ-thrombin was studied by monitoring the decrease in tryptophan fluorescence intensity of thrombin. We conclude that not only the N-terminal but also the electronegative aspartic residues D224, D230 and D235 (located in ECLII) are be important for PAR4 binding to thrombin. We further suggest that they play a role for the tethered ligand binding to the receptor, as mutations also affected activation in response to a PAR4-activating peptide mimicking the new N-terminal formed after cleavage. This agrees with previous results on PAR1 and thrombin binding. We suggest that the ECLII of PAR4 could be a potential target for antithrombotic drug development. Copyright © 2017 Elsevier Ltd. All rights reserved.

  11. Biomimetic L-aspartic acid-derived functional poly(ester amide)s for vascular tissue engineering.

    Science.gov (United States)

    Knight, Darryl K; Gillies, Elizabeth R; Mequanint, Kibret

    2014-08-01

    Functionalization of polymeric biomaterials permits the conjugation of cell signaling molecules capable of directing cell function. In this study, l-phenylalanine and l-aspartic acid were used to synthesize poly(ester amide)s (PEAs) with pendant carboxylic acid groups through an interfacial polycondensation approach. Human coronary artery smooth muscle cell (HCASMC) attachment, spreading and proliferation was observed on all PEA films. Vinculin expression at the cell periphery suggested that HCASMCs formed focal adhesions on the functional PEAs, while the absence of smooth muscle α-actin (SMαA) expression implied the cells adopted a proliferative phenotype. The PEAs were also electrospun to yield nanoscale three-dimensional (3-D) scaffolds with average fiber diameters ranging from 130 to 294nm. Immunoblotting studies suggested a potential increase in SMαA and calponin expression from HCASMCs cultured on 3-D fibrous scaffolds when compared to 2-D films. X-ray photoelectron spectroscopy and immunofluorescence demonstrated the conjugation of transforming growth factor-β1 to the surface of the functional PEA through the pendant carboxylic acid groups. Taken together, this study demonstrates that PEAs containing aspartic acid are viable biomaterials for further investigation in vascular tissue engineering. Copyright © 2014 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.

  12. Poly(aspartic acid) with adjustable pH-dependent solubility.

    Science.gov (United States)

    Németh, Csaba; Gyarmati, Benjámin; Abdullin, Timur; László, Krisztina; Szilágyi, András

    2017-02-01

    Poly(aspartic acid) (PASP) derivatives with adjustable pH-dependent solubility were synthesized and characterized to establish the relationship between their structure and solubility in order to predict their applicability as a basic material for enteric coatings. Polysuccinimide, the precursor of PASP, was modified with short chain alkylamines, and the residual succinimide rings were subsequently opened to prepare the corresponding PASP derivatives. Study of the effect of the type and concentration of the side groups on the pH-dependent solubility of PASP showed that solubility can be adjusted by proper selection of the chemical structure. The Henderson-Hasselbalch (HH) and the extended HH equations were used to describe the pH-dependent solubility of the polymers quantitatively. The estimate provided by the HH equation is poor, but an accurate description of the pH-dependent solubility can be found with the extended HH equation. The dissolution rate of a polymer film prepared from a selected PASP derivative was determined by fluorescence marking. The film dissolved rapidly when the pH was increased above its pK a . Cellular viability tests show that PASP derivatives are non-toxic to a human cell line. These polymers are thus of great interest as starting materials for enteric coatings. Poly(amino acid) type biocompatible polymers were synthesized for future use as pharmaceutical film coatings. To this end, we tailored the pH-dependent solubility of poly(aspartic acid) (PASP). It was found that both the solubility and the pK a values of the modified PASP depended strongly on composition. Fluorescent marking was used to characterize the dissolution of a chosen PASP derivative. In acidic media only a negligible amount of the polymer dissolved, but dissolution was very fast and complete at the pH values that prevail in the small intestine. As a consequence, enteric coatings based on such PASP derivatives may be used for drug delivery in the gastrointestinal tract

  13. Gender differences in D-aspartic acid content in skull bone.

    Science.gov (United States)

    Torikoshi-Hatano, Aiko; Namera, Akira; Shiraishi, Hiroaki; Arima, Yousuke; Toubou, Hirokazu; Ezaki, Jiro; Morikawa, Masami; Nagao, Masataka

    2012-12-01

    In forensic medicine, the personal identification of cadavers is one of the most important tasks. One method of estimating age at death relies on the high correlation between racemization rates in teeth and actual age, and this method has been applied successfully in forensic odontology for several years. In this study, we attempt to facilitate the analysis of racemized amino acids and examine the determination of age at death on the basis of the extent of aspartic acid (Asp) racemization in skull bones. The specimens were obtained from 61 human skull bones (19 females and 42 males) that underwent judicial autopsy from October 2010 to May 2012. The amount of D-Asp and L-Asp, total protein, osteocalcin, and collagen I in the skull bones was measured. Logistic regression analysis was performed for age, sex, and each measured protein. The amount of D-Asp in the female skull bones was significantly different from that in the male skull bones (p = 0.021), whereas the amount of L-Asp was similar. Thus, our study indicates that the amount of D-Asp in skull bones is different between the sexes.

  14. Anti-N-methyl-D-aspartate receptor encephalitis: three cases report and review of literature

    Directory of Open Access Journals (Sweden)

    Guan-en ZHOU

    2014-07-01

    Full Text Available Objective To study the clinical and laboratory features and diagnosis of the patient with anti-N-methyl-D-aspartate receptor (NMDAR encephalitis.  Methods The data of clinical features, laboratory findings, and radiological manifestations of 3 patients with anti-NMDAR encephalitis were reviewed and analyzed. Results Of the 3 patients, 2 were male and one was female. The age was from 33 to 34 years (33.30 years on average. Main symptoms included headache in 2 cases, psychiatric symptoms and speech disorder in 3 cases, different levels of movement disorder in one case and hallucinations in one case. The results of MRI examination revealed gyri swelling, abnormal signal and demyelination of temporal lobe. The EEG showed focal or diffuse slow waves. All cases were confirmed to have the disease by detection of anti-NMDAR antibodies. Both the white blood cell count (3 cases and protein quantification (2 cases elevated. No tumor was detected in any of the patients. All patients were coued after receiving immunotherapy with methylprednisolone and human immunoglobulin.  Conclusions Anti-NMDAR encephalitis is a severe but treatable disorder. The syndrome is highly recognizable clinically and can be confirmed with the demonstration of anti-NMDAR antibodies. Timely diagnosis and treatment may yield a favorable prognosis. doi: 10.3969/j.issn.1672-6731.2014.07.005

  15. Proteome-level assessment of origin, prevalence and function of Leucine-Aspartic Acid (LD) motifs

    KAUST Repository

    Alam, Tanvir

    2018-03-11

    Short Linear Motifs (SLiMs) contribute to almost every cellular function by connecting appropriate protein partners. Accurate prediction of SLiMs is difficult due to their shortness and sequence degeneracy. Leucine-aspartic acid (LD) motifs are SLiMs that link paxillin family proteins to factors controlling (cancer) cell adhesion, motility and survival. The existence and importance of LD motifs beyond the paxillin family is poorly understood. To enable a proteome-wide assessment of these motifs, we developed an active-learning based framework that iteratively integrates computational predictions with experimental validation. Our analysis of the human proteome identified a dozen proteins that contain LD motifs, all being involved in cell adhesion and migration, and revealed a new type of inverse LD motif consensus. Our evolutionary analysis suggested that LD motif signalling originated in the common unicellular ancestor of opisthokonts and amoebozoa by co-opting nuclear export sequences. Inter-species comparison revealed a conserved LD signalling core, and reveals the emergence of species-specific adaptive connections, while maintaining a strong functional focus of the LD motif interactome. Collectively, our data elucidate the mechanisms underlying the origin and adaptation of an ancestral SLiM.

  16. Computational studies on non-succinimide-mediated stereoinversion mechanism of aspartic acid residues assisted by phosphate

    Science.gov (United States)

    Nakayoshi, Tomoki; Fukuyoshi, Shuichi; Takahashi, Ohgi; Oda, Akifumi

    2018-03-01

    Although nearly all of the amino acids that constitute proteins are l-amino acids, d-amino acid residues in human proteins have been recently reported. d-amino acid residues cause a change in the three-dimensional structure of proteins, and d-aspartic acid (Asp) residues are considered to be one of the causes of age-related diseases. The stereoinversion of Asp residues in peptides and proteins is thought to proceed via a succinimide intermediate; however, it has been reported that stereoinversion can occur even under conditions where a succinimide intermediate cannot be formed. In order to elucidate the non-succinimide-mediated stereoinversion pathway, we investigated the stereoinversion of l-Asp to d-Asp catalysed by phosphate and estimated the activation barrier using B3LYP/6-31+G(d,p) density functional theory (DFT) calculations. For the DFT calculations, a model compound in which the Asp residue is capped with acetyl and methyl-amino groups on the N- and C-termini, respectively, was used. The calculated activation barrier was not excessively high for the stereoinversion to occur in vivo. Therefore, this stereoinversion mechanism may compete with the succinimide-mediated mechanism.

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

    Indian Academy of Sciences (India)

    PRAKASH KUMAR

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

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

    African Journals Online (AJOL)

    AJB SERVER

    2006-10-19

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

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

    African Journals Online (AJOL)

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

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

    African Journals Online (AJOL)

    Erah

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