Czech Academy of Sciences Publication Activity Database
Dzyuba, V.; Słowińska, M.; Cosson, J.; Ciereszko, A.; Boryshpolets, S.; Štěrba, Ján; Rodina, M.; Linhart, O.; Dzyuba, B.
2016-01-01
Roč. 42, č. 6 (2016), s. 1755-1766 ISSN 0920-1742 R&D Projects: GA MŠk(CZ) EE2.3.30.0032 Institutional support: RVO:60077344 Keywords : amidase activity * anti-proteolytic activity * casein and gelatin zymography * siberian sturgeon sperm * spermatozoon maturation * sterlet sperm Subject RIV: ED - Physiology Impact factor: 1.647, year: 2016
Proteolytic activity of alkaliphilic, salt-tolerant actinomycetes from ...
African Journals Online (AJOL)
tolerant alkaliphilic. All the isolates need to be further studied for the ability of their potential protease enzyme production. Key words: Alkaliphilic actinomycetes, salt tolerant actinomycetes, desert soil, isolation, proteolytic activity.
New radionuclide method for determination of gastric content proteolytic activity. (Experiments)
International Nuclear Information System (INIS)
Sergienko, V.B.; Popova, L.V.; Khodarev, N.N.; Astashenkova, K.Yu.
1988-01-01
A possibility of the use of a radionuclide tubeless rapid method for measuring gastric content proteolytic activity (GCPA) with the help of a protein (gelatin) RP containing capsule was demonstrated in experiments in vitro. There was correlation between the time of dissolution of RP containing capsules and GCPA determined after Mett's method. Reference time intervals were established for normal, raised and lowered proteolytic activity. The method was shown to be physiological, simple and timesaving
Pinilla, Yudi T; Moreno-Pérez, Darwin A; Patarroyo, Manuel A; Bello, Felio J
2013-12-01
Sarconesiopsis magellanica (Diptera: Calliphoridae) is a medically important necrophagous fly which is used for establishing the post-mortem interval. Diptera maggots release proteolytic enzymes contained in larval excretion and secretion (ES) products playing a key role in digestion. Special interest in proteolytic enzymes has also been aroused regarding understanding their role in wound healing since they degrade necrotic tissue during larval therapy. This study was thus aimed at identifying and characterising S. magellanica proteolytic enzyme ES products for the first time. These products were obtained from first-, second- and third-instar larvae taken from a previously-established colony. ES proteins were separated by SDS-PAGE and their proteolytic activity was characterised by zymograms and inhibition assays involving BAPNA (Nα-benzoyl-dl-Arg-p-nitroanilide) and SAPNA substrates, using synthetic inhibitors. The protein profile ranged from ∼69kDa to ∼23kDa; several of them coincided with the Lucilia sericata ES protein profile. Serine-protease hydrolysis activity (measured by zymogram) was confirmed when a ∼25kDa band disappeared upon ES incubation with PMSF inhibitor at pH 7.8. Analysis of larval ES proteolytic activity on BAPNA and SAPNA substrates (determined by using TLCK and TPCK specific inhibitors) suggested a greater amount of trypsin-like protease. These results support the need for further experiments aimed at validating S. magellanica use in larval therapy. Copyright © 2013 Elsevier B.V. All rights reserved.
Brzostek, Edward R.; Finzi, Adrien C.
2012-03-01
Increasing soil temperature has the potential to alter the activity of the extracellular enzymes that mobilize nitrogen (N) from soil organic matter (SOM) and ultimately the availability of N for primary production. Proteolytic enzymes depolymerize N from proteinaceous components of SOM into amino acids, and their activity is a principal driver of the within-system cycle of soil N. The objectives of this study were to investigate whether the soils of temperate forest tree species differ in the temperature sensitivity of proteolytic enzyme activity over the growing season and the role of substrate limitation in regulating temperature sensitivity. Across species and sampling dates, proteolytic enzyme activity had relatively low sensitivity to temperature with a mean activation energy (Ea) of 33.5 kJ mol-1. Ea declined in white ash, American beech, and eastern hemlock soils across the growing season as soils warmed. By contrast, Eain sugar maple soil increased across the growing season. We used these data to develop a species-specific empirical model of proteolytic enzyme activity for the 2009 calendar year and studied the interactive effects of soil temperature (ambient or +5°C) and substrate limitation (ambient or elevated protein) on enzyme activity. Declines in substrate limitation had a larger single-factor effect on proteolytic enzyme activity than temperature, particularly in the spring. There was, however, a large synergistic effect of increasing temperature and substrate supply on proteolytic enzyme activity. Our results suggest limited increases in N availability with climate warming unless there is a parallel increase in the availability of protein substrates.
Proteolytic and antimicrobial activity of lactic acid bacteria grown in goat milk.
Atanasova, Jivka; Moncheva, Penka; Ivanova, Iskra
2014-11-02
We examined 62 strains and 21 trade starter cultures from the collection of LB Bulgaricum PLC for proteolytic and antimicrobial activity of lactic acid bacteria (LAB) grown in goat milk. The aim of this study was to investigate the fermentation of caseins, α-lactalbumin and β-lactoglobulin by LAB, using the o -phthaldialdehyde (OPA) spectrophotometric assay and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The proteolysis targeted mainly caseins, especially β-casein. Whey proteins were proteolyzed, essentially β-lactoglobulin. The proteolytic activity of Lactococcus lactis l598, Streptococcus thermophilus t3D1, Dt1, Lactobacillus lactis 1043 and L. delbrueckii subsp. bulgaricus b38, b122 and b24 was notably high. The proteolysis process gave rise to medium-sized peptide populations. Most of the examined strains showed antimicrobial activity against some food pathogens, such as Escherichia coli , Staphylococcus aureus , Salmonella cholere enteridis , Listeria monocytogenes , Listeria innocua and Enterobacter aerogenes . The most active producers of antimicrobial-active peptides were strains of L. delbrueckii subsp. bulgaricus and S. thermophilus , which are of practical importance. The starter cultures containing the examined species showed high proteolytic and antimicrobial activity in skimmed goat milk. The greatest antimicrobial activity of the cultures was detected against E. aerogenes . The obtained results demonstrated the significant proteolytic potential of the examined strains in goat milk and their potential for application in the production of dairy products from goat's milk. The present results could be considered as the first data on the proteolytic capacity of strains and starter cultures in goat milk for the purposes of trade interest of LB Bulgaricum PLC.
[Proteolytic activity of IgG-antibodies of mice, immunized by calf thymus histones].
Kit, Iu Ia; Korniĭ, N; Kril', I Ĭ; Mahorivs'ka, I B; Tkachenko, V; Bilyĭ, R O; Stoĭka, R S
2014-01-01
The main goal of the study was to determine the ability of histones to induce production of the proteolytically active IgG-antibodies in BALB/c mice. In order to perform this study 8 mice were immunized with the fraction of total calf thymus histones. IgGs were isolated from the serum of the immunized and not immunized animals by means of precipitation with 33% ammonium sulfate, followed by affinity chromatography on protein G-Sepharose column. Histones, myelin basic protein (MBP), lysozyme, BSA, ovalbumin, macroglobulin, casein and cytochrome c served as substrates for determining the proteolytic activity. It was found that IgGs from the blood serum of immunized mice are capable of hydrolyzing histone H1, core histone and MBP. On the contrary, the proteolytic activity of IgGs from the blood serum of not immunized mice was not detected. The absence of proteolytical enzymes in the fraction of IgGs was proven by HPLC chromatography. High levels of proteolytic activity toward histones have been also detected in affinity purified IgGs from blood serum of patients with rheumatoid arthritis, but not in healthy donors. These data indicate that eukaryotic histones may induce production of protabzymes in mammals. The possible origin of these protabzymes and their potential biological role in mammalians is discussed.
Optical Imaging of Matrix Metalloproteinase-7 Activity in Vivo Using a Proteolytic Nanobeacon
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Randy L. Scherer
2008-05-01
Full Text Available Matrix metalloproteinases (MMPs are extracellular proteolytic enzymes involved in tumor progression. We present the in vivo detection and quantitation of MMP7 activity using a specific near-infrared polymer-based proteolytic beacon, PB-M7NIR. PB-M7NIR is a pegylated polyamidoamine PAMAM-Generation 4 dendrimer core covalently coupled to a Cy5.5-labeled peptide representing a selective substrate that monitors MMP7 activity (sensor and AF750 as an internal reference to monitor relative substrate concentration (reference. In vivo imaging of tumors expressing MMP7 had a median sensor to reference ratio 2.2-fold higher than a that of a bilateral control tumor. Ex vivo imaging of intestines of multiple intestinal neoplasia (APCMin mice injected systemically with PB-M7NIR revealed a sixfold increase in the sensor to reference ratio in the adenomas of APCMin mice compared with control intestinal tissue or adenomas from MMP7-null Min mice. PB-M7NIR detected tumor sizes as small as 0.01 cm2, and the sensor to reference ratio was independent of tumor size. Histologic sectioning of xenograft tumors localized the proteolytic signal to the extracellular matrix; MMP7-overexpressing tumors displayed an approximately 300-fold enhancement in the sensor to reference ratio compared with nonexpressing tumor cells. In APCMin adenomas, the proteolytic signal colocalized with the endogenously expressed MMP7 protein, with sensor to reference ratios approximately sixfold greater than that of normal intestinal epithelium. PB-M7NIR provides a useful reagent for the in vivo and ex vivo quantitation and localization of MMP-selective proteolytic activity.
Wing, S. S.; Goldberg, A. L.; Goldberger, A. L. (Principal Investigator)
1993-01-01
Glucocorticoids are essential for the increase in protein breakdown in skeletal muscle normally seen during fasting. To determine which proteolytic pathway(s) are activated upon fasting, leg muscles from fed and fasted normal rats were incubated under conditions that block or activate different proteolytic systems. After food deprivation (1 day), the nonlysosomal ATP-dependent process increased by 250%, as shown in experiments involving depletion of muscle ATP. Also, the maximal capacity of the lysosomal process increased 60-100%, but no changes occurred in the Ca(2+)-dependent or the residual energy-independent proteolytic processes. In muscles from fasted normal and adrenalectomized (ADX) rats, the protein breakdown sensitive to inhibitors of the lysosomal or Ca(2+)-dependent pathways did not differ. However, the ATP-dependent process was 30% slower in muscles from fasted ADX rats. Administering dexamethasone to these animals or incubating their muscles with dexamethasone reversed this defect. During fasting, when the ATP-dependent process rises, muscles show a two- to threefold increase in levels of ubiquitin (Ub) mRNA. However, muscles of ADX animals failed to show this response. Injecting dexamethasone into the fasted ADX animals increased muscle Ub mRNA within 6 h. Thus glucocorticoids activate the ATP-Ub-dependent proteolytic pathway in fasting apparently by enhancing the expression of components of this system such as Ub.
Investigation of plant latices of Asteraceae and Campanulaceae regarding proteolytic activity.
Sytwala, Sonja; Domsalla, André; Melzig, Matthias F
2015-12-01
Occurrence of plant latices is widespread, there are more than 40 families of plants characterized to establish lactiferous structures. The appearance of hydrolytic active proteins, incorporated in latices is already characterized, and hydrolytic active proteins are considerable, and for several plant families, the occurrence of hydrolytic active proteins is already specified e.g. Apocynaceae Juss., Caricaceae Dumort, Euphorbiaceae Juss., Moraceae Gaudich and Papaveraceae Juss. In our investigation, focused on latex bearing plants of order Asterales, Asteraceae and Campanulaceae in particular. The present outcomes represent a comprehensive study, relating to the occurrence of proteolytic active enzymes of order Asterales for the first time. 131 different species of Asteraceae and Campanulaceae were tested, and the appearance of plant latex proteases were determined in different quantities. Proteolytic activity was investigated by inhibitory studies and determination of residual activity in the following, enable us to characterize the proteases. Most of the considered species exhibit a serine protease activity and a multiplicity of species exhibited two or more subclasses of proteases. Copyright © 2015 Elsevier Masson SAS. All rights reserved.
Mucha, Joanna; Dahm, Hanna; Werner, Antoni
2007-07-01
The production of proteolytic enzymes by several strains of ectomycorrhizal fungi i.e., Amanita muscaria (16-3), Laccaria laccata (9-12), L. laccata (9-1), Suillus bovinus (15-4), Suillus bovinus (15-3), Suillus luteus (14-7) on mycelia of Trichoderma harzianum, Trichoderma virens and Mucor hiemalis and sodium caseinate, yeast extract was evaluated. The strains of A. muscaria (16-3) and L. laccata (9-12) were characterized by the highest activity of the acidic and neutral proteases. Taking the mycelia of saprotrophic fungi into consideration, the mycelium of M. hiemalis was the best inductor for proteolytic activity. The examined ectomycorrhizal fungi exhibited higher activity of acidic proteases than neutral ones on the mycelia of saprotrophic fungi, which may imply the participation of acidic proteases in nutrition.
Determination of proteolytic activity using L-[4,5-3H]leucine-labelled globin as a substrate
International Nuclear Information System (INIS)
Maliopoulou, T.B.; Dionyssiou-Asteriou, A.; Loucopoulos, D.
1980-01-01
A simple and sensitive method for the assay of proteolytic enzyme activity is described. This is based on the digestion of L-[4,5- 3 H]leucine globin by proteolytic enzymes and radioactivity measurement of the trichloroacetic acid soluble cleavage products. (Auth.)
Pakshir, K; Mohamadi, T; Khodadadi, H; Motamedifar, M; Zomorodian, K; Alipour, S; Motamedi, M
2016-01-01
Background and Purpose: Globally, dermatophytes are the most common filamentous group of fungi causing cutaneous mycoses. Dermatophytes were shown to secrete a multitude of enzymes that play a role in their pathogenesis. There is limited data on co-hemolytic (CAMP-like) effect of different bacterial species on dermatophyte species. In this study, we sought to the evaluate exoenzyme activity and co-hemolytic effect of four bacteria on clinical dermatophytes isolated from patients in Shiraz, Iran. Materials and Methods: A total of 84 clinical dermatophyte species were isolated from patients suffering dermatophytosis and identified by conventional methods. Hemolytic activity was evaluated with Columbia 5% sheep blood agar. Proteolytic activity was determined by plate clearance assay method, using gelatin 8% agar. CAMP-like factor was evaluated with four bacteria, namely, S. areus, S. saprophyticus, S. pyogenes, and S. agalactiae. Fisher's exact test was run for statistical analysis. Results: T. mentagrophytes was the most predominant agent (27 [32.1%]) followed by T. verrucosum(20 [23.8%]), T. tonsurans (10 [11.9%]), Microsporum canis (7 [8.3%]), T. rubrum (6 [7.1%]), E. floccosum (6 [7.1%]), M. gypseum (5 [6%]), and T. violaceum (3[3.6%]). The most common clinical area of dermatophytosis was the skin. All the isolates expressed the zone of incomplete alpha hemolysis. All the isolates had CAMP- positive reaction with S. aureus and the other bacteria were CAMP-negative. All the isolates expressed proteolytic activity and no significant differences were noted among diverse genera of dermatophytes and severities of proteolytic activity. Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species. PMID:28959790
Proteolytic activity of IgGs from blood serum of wistar rats at experimental rheumatoid arthritis
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Yu. Ya. Kit
2014-10-01
Full Text Available The aim of this work was to study the proteolytic activity of IgGs purified from blood serum of Wistar rats at experimental rheumatoid arthritis (ERA induced by an injection of bovine collagen of type II. Twenty rats were immunized with a preparation of bovine collagen II (Sigma-Aldrich, USA in the presence of complete Freund’s adjuvant. ERA development was determined by inflammation in limbs of treated animals. IgG preparations were isolated from blood serum of immunized and non-immunized animals by precipitation of antibodies with 33% ammonium sulfate followed by chromatography on the Protein G-Sepharose column. Human histone H1, bovine collagen II, calf thymus histones, myelin basic protein (MBP, bovine serum albumin (BSA, and bovine casein were used as substrates of the proteolytic activity of IgGs. It was found that IgG preparations from blood serum of rats with ERA were capable of cleaving histone H1 and MBP, however, they were catalytically inactive towards collagen II, casein, BSA, and core histones. IgGs from blood serum of non-immunized rats were proteolytically inactive towards all used protein substrates. Thus, we demonstrated that immunization of rats with bovine collagen II induced IgG-antibodies possessing the proteolytic activity towards histone H1 and MBP. This activity might be associated with the development of inflammatory processes in the immunized rats.
Proteolytic activity of recombinant DegP from Chromohalobacter salexigens BKL5
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Dewi Fitriani
2017-09-01
Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when β-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.
Mattsson, Johanna M.; Ravela, Suvi; Hekim, Can; Jonsson, Magnus; Malm, Johan; Närvänen, Ale; Stenman, Ulf-Håkan; Koistinen, Hannu
2014-01-01
Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3) exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA. PMID:25237904
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Johanna M Mattsson
Full Text Available Prostate-specific antigen (PSA or kallikrein-related peptidase-3, KLK3 exerts chymotrypsin-like proteolytic activity. The main biological function of PSA is the liquefaction of the clot formed after ejaculation by cleavage of semenogelins I and II in seminal fluid. PSA also cleaves several other substrates, which may explain its putative functions in prostate cancer and its antiangiogenic activity. We compared the proteolytic efficiency of PSA towards several protein and peptide substrates and studied the effect of peptides stimulating the activity of PSA with these substrates. An endothelial cell tube formation model was used to analyze the effect of PSA-degraded protein fragments on angiogenesis. We showed that PSA degrades semenogelins I and II much more efficiently than other previously identified protein substrates, e.g., fibronectin, galectin-3 and IGFBP-3. We identified nidogen-1 as a new substrate for PSA. Peptides B2 and C4 that stimulate the activity of PSA towards small peptide substrates also enhanced the proteolytic activity of PSA towards protein substrates. Nidogen-1, galectin-3 or their fragments produced by PSA did not have any effect on endothelial cell tube formation. Although PSA cleaves several other protein substrates, in addition to semenogelins, the physiological importance of this activity remains speculative. The PSA levels in prostate are very high, but several other highly active proteases, such as hK2 and trypsin, are also expressed in the prostate and may cleave protein substrates that are weakly cleaved by PSA.
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Asep A. Prihanto
2013-04-01
Full Text Available Exploration of fermented foods as sources of fibrinolytic enzymes is increased in the last decades. Terasi and Jambal roti is Indonesian traditional fermented fish products, which were famous in Java Island. Both are important products in Indonesian dishes, especially in Java. Investigation on halophilic lactic acid bacteria using MRS and M-17 agar obtained seventy four isolated strains. Their proteolytic and fibrinolytic activities were determined using skim milk agar and plasminogen-free fibrin plate. Twenty five isolates showed protease activities, while only four of them secreted fibrinolitic enzyme. The highest proteolytic and fibrinolytic activity was shown by TB1 strain, which is identified as Bacillus coagulans. The 16s rDNA is still in investigating to confirm the TB1 strain identity.
Tremonte, P; Reale, A; Di Renzo, T; Tipaldi, L; Di Luccia, A; Coppola, R; Sorrentino, E; Succi, M
2010-11-01
To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell-free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures. © 2010 The Authors. © 2010 The Society for Applied Microbiology.
Proteolytic activities in yeast after UV irradiation. Pt. 1
International Nuclear Information System (INIS)
Schwencke, J.; Moustacchi, E.
1982-01-01
Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD + yeast cells after a dose of 50 Jm -2 of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD + strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented. (orig.)
Proteolytic activities in yeast after UV irradiation. Pt. 1
Energy Technology Data Exchange (ETDEWEB)
Schwencke, J.; Moustacchi, E.
1982-04-01
Specific proteolytic activities are known to be induced in Escherichia coli following irradiation. Consequently it seemed of interest to investigate whether variations in proteinase activities occur in yeast. Among the five most well known proteinases of Saccharomyces cerevisiae, we have found that proteinase B activity increases up to three times in wild-type RAD/sup +/ yeast cells after a dose of 50 Jm/sup -2/ of 254 nm ultraviolet light (40% survival). Carboxypeptidase Y and aminopeptidase I (leucin aminopeptidase) activities were only moderately increased. Proteinase A activity was only slightly enhanced, while aminopeptidase II (lysin aminopeptidase) was unaffected in both RAD/sup +/ strains studied. The observed post UV-increase in proteinase B activity was inhibited by cycloheximide and was dose dependent. Increases in proteinase B levels were independent of the activation method used to destroy the proteinase B-inhibitor complex present in the crude yeast extracts. A standard method for comparison of the postirradiation levels among different proteinases, strains and methods of activation is presented.
Energy Technology Data Exchange (ETDEWEB)
Blumentals, I.I.; Robinson, A.S.; Kelly, R.M. (Johns Hopkins Univ., Baltimore, MD (USA))
1990-07-01
Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98{degree}C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons (kDa)) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98{degree}C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% {beta}-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis({beta}-aminoethyl ether)-N,N,N{prime},N{prime}-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.
Proteolytic Activity in Reduced-Fat Cheddar Cheese Made with Lactic Acid Bacteria and Camel Chymosin
DEFF Research Database (Denmark)
Børsting, Mette Winther
be the need of an extended ripening period to reach a similar cheese structure as in cheeses produced with BC. The aim of this project was to compensate for the lower proteolytic activity in cheese produced with CC compared to BC. Selection of dairy lactic acid bacteria (LAB) for cheese production with high....... lactis subsp lactis, 10 thermophilic Lactobacillus strains and 15 frozen direct vat set strains of thermopholic Lactobacillus) to hydrolyse αS1-CN, candidates were selected for cheese-making experiments. None of the selected proteolytic strains contributed significantly to softening the cheese structure...
Caffrey, C R; Ryan, M F
1994-04-01
An excretory-secretory (ES) preparation derived from adult Strongylus vulgaris in vitro was assessed for proteolytic activity using azocasein and synthetic, fluorogenic, peptide substrates. Fractionation was by molecular sieve fast protein liquid chromatography (molecular sieve FPLC) and resolution by gelatin-substrate sodium dodecyl sulphate-polyacrylamide gel electrophoresis (gelatin-substrate SDS-PAGE). The cysteine proteinase activator, dithiothreitol (DTT), enhanced azocaseinolysis and hydrolysis of carbobenzoxy-phenylalanyl-arginine-7-amido-4-methylcoumarin (Z-Phe-Arg-NMec) by the ES preparation and was a requirement for the detection of carbobenzoxy-arginyl-arginine-7-amido-4-methylcoumarin (Z-Arg-Arg-NMec) hydrolysis. Assays of FPLC-eluted fractions, with DTT, detected a broad peak of azocaseinolytic activity (22-24 kDa) and two peaks (24 and 18 kDa) of hydrolysis using the synthetic substrates. Hydrolysis by these peaks of Z-Phe-Arg-NMec was 50-fold greater than that of Z-Arg-Arg-NMec suggesting that their specificities are more like papain or cathepsin L rather than cathepsin B. In gelatin-substrate SDS-PAGE, DTT was required to detect proteolysis by the ES preparation which was optimal at pH 6.0 and resolved into eight bands (87-29 kDa). Cysteine proteinase inhibitors were the most effective in all assays. Collectively, these data indicate that cysteine-class proteolytic activity predominates in the ES preparation of adult S. vulgaris.
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Fediv A.I. Fediv A.I.
2016-09-01
Full Text Available Aim of the study — to conduct the analysis of the performance of proteolytic and fibrinolytic activity of the blood plasma in patients with bronchial asthma (BA combined with obesity. Materials and methods. The study included 40 patients divided into groups. The basic group consisted of 20 patients with BA associated with obesity (ІI group, two comparison groups — of 10 patients with BA and normal body weight (I group and 10 patients with obesity and without pathology of the bronchopulmonary system (ІІІ group. Control group included 10 apparently healthy persons. In patients with BA, there were investigated the indexes of proteolytic and fibrinolytic activity of the blood plasma. Results. Analysis of the obtained data in patients with BA showed an increase of activity of plasma coagulation factors: total fibrinolytic activity increased in comparison to the control group, and in patients with BA it was 1.46 ± 0.13 Е440/ml/h, while in apparently healthy persons — 1.23 ± 0.16 Е440/ml/h (р < 0.05. In addition, an increase was marked and in the group of patients with the isolated obesity (by 38.5 % as compared to the group of apparently healthy persons, that can be explained by the growth of activity of inflammatory process due to the immune changes related to biological activity of fatty tissue as an additional source of proinflammatory cytokines. Conclusions. In combination of bronchial asthma and obesity, there is an activation of fibrinolytic and proteolytic systems of the blood that leads to microcirculatory and hemostatic disorders, enhancement of inflammatory process. In obesity as the state of systemic inflammatory response, the activation of fibrinolytic and proteolytic blood systems is more significant, than in bronchial asthma.
Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.
Zhao, Luping; Kong, Xiangzhen; Zhang, Caimeng; Hua, Yufei; Chen, Yeming
2017-07-19
P34 probable thiol protease (P34) and Gly m Bd 30K (30K) show high relationship with the protease of 24 kDa oleosin of soybean oil bodies. In this study, 9 day germinated soybean was used to separate bioprocessed P34 (P32) from bioprocessed 30K (28K). Interestingly, P32 existed as dimer, whereas 28K existed as monomer; a P32-rich sample had proteolytic activity and high cleavage site specificity (Lys-Thr of 24 kDa oleosin), whereas a 28K-rich sample showed low proteolytic activity; the P32-rich sample contained one thiol protease. After mixing with purified oil bodies, all P32 dimers were dissociated and bound to 24 kDa oleosins to form P32-24 kDa oleosin complexes. By incubation, 24 kDa oleosin was preferentially hydrolyzed, and two hydrolyzed products (HPs; 17 and 7 kDa) were confirmed. After most of 24 kDa oleosin was hydrolyzed, some P32 existed as dimer, and the other as P32-17 kDa HP. It was suggested that P32 was the protease.
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Yusmarini1,2*
2010-12-01
Full Text Available Some lactic acid bacteria (LAB strains had been isolated from spontaneously fermented soymilk which have proteolytic system. The purpose of this research was to study ability of isolates in fermentation of soymilk. The changes in bacterial growth, pH, titrable acidity, and proteolytic activities during fermentation were examined. Isolates of Lactobacillus plantarum 1 R.1.3.2; L. plantarum 1 R.11.1.2 and L. acidophilus FNCC 0051 (as a control were capable growing in soymilk. The results indicated that initial pH of soymilk was 6,6 and decreased to 4,6 after fermentation and titrable acidity of 0.11 increased to 0.34 after fermentation. The proteolytic activities were 0.352 U/ml – 0.468 U/ml. The electrophoretic pattern of the proteins showed changes during fermentation of soymilk.
International Nuclear Information System (INIS)
Toema, M.A.; El-Bazza, Z.E.; El-Hifnawi, H.N.; Abd-El-Hakim, E.E.
2013-01-01
In this study, clinical samples were collected from 100 patients admitted to Burn and Plastic Surgery Department, Faculty of Medicine, Ain Shams University, Egypt, over a period of 12 months. The proteolytic activity of 110 clinical samples taken from surfaces swabs which taken from burned and burned wounded patients with different ages and gender was examined. Screening for the proteolytic activity produced by pathogenic bacteria isolated from burned and burned wounded patients was evaluated as gram positive Bacilli and gram negative bacilli showed high proteolytic activity (46.4%) while 17.9% showed no activity. The isolated bacteria proved to have proteolytic activity were classified into high, moderate and weak. The pathogenic bacteria isolated from burned and burned wounded patients and showing proteolytic activity were identified as Pseudomonas aeruginosa, Proteus mirabilis, Proteus vulgaris, Bacillus megaterium, Bacillus cereus, Staphylococcus aureus, Escherichia coli, Klebsiella ozaeanae, Klebsiella oxytoca, Klebsiella pneumoniae and Pseudomonas fluoresces.
Briens, Aurélien; Gauberti, Maxime; Parcq, Jérôme; Montaner, Joan; Vivien, Denis; Martinez de Lizarrondo, Sara
2016-01-01
Cell-derived microparticles (MPs) are nano-sized vesicles released by activated cells in the extracellular milieu. They act as vectors of biological activity by carrying membrane-anchored and cytoplasmic constituents of the parental cells. Although detection and characterization of cell-derived MPs may be of high diagnostic and prognostic values in a number of human diseases, reliable measurement of their size, number and biological activity still remains challenging using currently available methods. In the present study, we developed a protocol to directly image and functionally characterize MPs using high-resolution laser-scanning confocal microscopy. Once trapped on annexin-V coated micro-wells, we developed several assays using fluorescent reporters to measure their size, detect membrane antigens and evaluate proteolytic activity (nano-zymography). In particular, we demonstrated the applicability and specificity of this method to detect antigens and proteolytic activities of tissue-type plasminogen activator (tPA), urokinase and plasmin at the surface of engineered MPs from transfected cell-lines. Furthermore, we were able to identify a subset of tPA-bearing fibrinolytic MPs using plasma samples from a cohort of ischemic stroke patients who received thrombolytic therapy and in an experimental model of thrombin-induced ischemic stroke in mice. Overall, this method is promising for functional characterization of cell-derived MPs.
Antifungal and proteolytic activities of endophytic fungi isolated from Piper hispidum Sw
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Ravely Casarotti Orlandelli
2015-06-01
Full Text Available Endophytes are being considered for use in biological control, and the enzymes they secrete might facilitate their initial colonization of internal plant tissues and direct interactions with microbial pathogens. Microbial proteases are also biotechnologically important products employed in bioremediation processes, cosmetics, and the pharmaceutical, photographic and food industries. In the present study, we evaluated antagonism and competitive interactions between 98 fungal endophytes and Alternaria alternata, Colletotrichum sp., Phyllosticta citricarpa and Moniliophthora perniciosa. We also examined the proteolytic activities of endophytes grown in liquid medium and conducted cup plate assays. The results showed that certain strains in the assemblage of P. hispidum endophytes are important sources of antifungal properties, primarily Lasiodiplodia theobromae JF766989, which reduced phytopathogen growth by approximately 54 to 65%. We detected 28 endophytes producing enzymatic halos of up to 16.40 mm in diameter. The results obtained in the present study highlight the proteolytic activity of the endophytes Phoma herbarum JF766995 and Schizophyllum commune JF766994, which presented the highest enzymatic halo diameters under at least one culture condition tested. The increased activities of certain isolates in the presence of rice or soy flour as a substrate (with halos up to 17.67 mm in diameter suggests that these endophytes have the potential to produce enzymes using agricultural wastes.
Activities of indigenous proteolytic enzymes in caprine milk of different somatic cell counts.
Albenzio, M; Santillo, A; Kelly, A L; Caroprese, M; Marino, R; Sevi, A
2015-11-01
Individual caprine milk with different somatic cell counts (SCC) were studied with the aim of investigating the percentage distribution of leukocyte cell types and the activities of indigenous proteolytic enzymes; proteolysis of casein was also studied in relation to cell type following recovery from milk. The experiment was conducted on 5 intensively managed dairy flocks of Garganica goats; on the basis of SCC, the experimental groups were denoted low (L-SCC; 1,501,000 cells/mL) SCC. Leukocyte distribution differed between groups; polymorphonuclear neutrophilic leukocytes were higher in M-SCC and H-SCC milk samples, the percentage macrophages was the highest in H-SCC, and levels of nonviable cells significantly decreased with increasing SCC. Activities of all the main proteolytic enzymes were affected by SCC; plasmin activity was the highest in H-SCC milk and the lowest in L-SCC, and elastase and cathepsin D activities were the highest in M-SCC. Somatic cell count influenced casein hydrolysis patterns, with less intact α- and β-casein in H-SCC milk. Higher levels of low electrophoretic mobility peptides were detected in sodium caseinate incubated with leukocytes isolated from L-SCC milk, independent of cell type, whereas among cells recovered from M-SCC milk, macrophages yielded the highest levels of low electrophoretic mobility peptides from sodium caseinate. The level of high electrophoretic mobility peptides was higher in sodium caseinate incubated with polymorphonuclear neutrophilic leukocytes and macrophages isolated from M-SCC, whereas the same fraction of peptides was always the highest, independent of leukocyte type, for cells recovered from H-SCC milk. In caprine milk, a level of 700,000 cells/mL represented the threshold for changes in leukocyte distribution, which is presumably related to the immune status of the mammary gland. Differences in the profile of indigenous lysosomal proteolytic enzymes in caprine milk may influence the integrity of casein
Biological and Proteolytic Variation in the Venom of Crotalus scutulatus scutulatus from Mexico
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Miguel Borja
2018-01-01
Full Text Available Rattlesnake venoms may be classified according to the presence/absence and relative abundance of the neurotoxic phospholipases A 2 s (PLA 2 s, such as Mojave toxin, and snake venom metalloproteinases (SVMPs. In Mexico, studies to determine venom variation in Mojave Rattlesnakes (Crotalus scutulatus scutulatus are limited and little is known about the biological and proteolytic activities in this species. Tissue (34 and venom (29 samples were obtained from C. s. scutulatus from different locations within their distribution in Mexico. Mojave toxin detection was carried out at the genomic (by PCR and protein (by ELISA levels for all tissue and venom samples. Biological activity was tested on representative venoms by measuring LD 50 and hemorrhagic activity. To determine the approximate amount of SVMPs, 15 venoms were separated by RP-HPLC and variation in protein profile and proteolytic activity was evaluated by SDS-PAGE (n = 28 and Hide Powder Azure proteolytic analysis (n = 27. Three types of venom were identified in Mexico which is comparable to the intraspecific venom diversity observed in the Sonoran Desert of Arizona, USA: Venom Type A (∼Type II, with Mojave toxin, highly toxic, lacking hemorrhagic activity, and with scarce proteolytic activity; Type B (∼Type I, without Mojave toxin, less toxic than Type A, highly hemorrhagic and proteolytic; and Type A + B, containing Mojave toxin, as toxic as venom Type A, variable in hemorrhagic activity and with intermediate proteolytic activity. We also detected a positive correlation between SVMP abundance and hemorrhagic and proteolytic activities. Although more sampling is necessary, our results suggest that venoms containing Mojave toxin and venom lacking this toxin are distributed in the northwest and southeast portions of the distribution in Mexico, respectively, while an intergradation in the middle of both zones is present.
Activation of human monocytes by proteolytic fragments of gliadin
Czech Academy of Sciences Publication Activity Database
Jelínková, Lenka; Tučková, Ludmila; Cinová, Jana; Cimburek, Zdeněk; Tlaskalová, Helena
2003-01-01
Roč. 87, č. 1 (2003), s. 08 ISSN 0165-2478 Institutional research plan: CEZ:AV0Z5020903 Keywords : proteolytic * fragments * gliadin Subject RIV: EE - Microbiology, Virology Impact factor: 1.710, year: 2003
Directory of Open Access Journals (Sweden)
Wiltrud Haaß
Full Text Available ESPL1/Separase, an endopeptidase, is required for centrosome duplication and separation of sister-chromatides in anaphase of mitosis. Overexpression and deregulated proteolytic activity of Separase as frequently observed in human cancers is associated with the occurrence of supernumerary centrosomes, chromosomal missegregation and aneuploidy. Recently, we have hypothesized that increased Separase proteolytic activity in a small subpopulation of tumor cells may serve as driver of tumor heterogeneity and clonal evolution in chronic myeloid leukemia (CML. Currently, there is no quantitative assay to measure Separase activity levels in single cells. Therefore, we have designed a flow cytometry-based assay that utilizes a Cy5- and rhodamine 110 (Rh110-biconjugated Rad21 cleavage site peptide ([Cy5-D-R-E-I-M-R]2-Rh110 as smart probe and intracellular substrate for detection of Separase enzyme activity in living cells. As measured by Cy5 fluorescence the cellular uptake of the fluorogenic peptide was fast and reached saturation after 210 min of incubation in human histiocytic lymphoma U937 cells. Separase activity was recorded as the intensity of Rh110 fluorescence released after intracellular peptide cleavage providing a linear signal gain within a 90-180 min time slot. Compared to conventional cell extract-based methods the flow cytometric assay delivers equivalent results but is more reliable, bypasses the problem of vague loading controls and unspecific proteolysis associated with whole cell extracts. Especially suited for the investigaton of blood- and bone marrow-derived hematopoietic cells the flow cytometric Separase assay allows generation of Separase activity profiles that tell about the number of Separase positive cells within a sample i.e. cells that currently progress through mitosis and about the range of intercellular variation in Separase activity levels within a cell population. The assay was used to quantify Separase proteolytic
DEFF Research Database (Denmark)
Kjær-Sørensen, Kasper; Engholm, Ditte Høyer; Kamei, Hiroyasu
2013-01-01
the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside the Igf system. Biochemical analysis of recombinant zebrafish...... Papp-a demonstrates conservation of proteolytic activity, specificity, and intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficient as wild-type Papp-a. Thus, the developmental...
Urinary Proteolytic Activation of Renal Epithelial Na+ Channels in Chronic Heart Failure.
Zheng, Hong; Liu, Xuefei; Sharma, Neeru M; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P
2016-01-01
One of the key mechanisms involved in renal Na(+) retention in chronic heart failure (CHF) is activation of epithelial Na(+) channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC, resulting in renal Na(+) retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared with sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06, and plasmin 3.57 versus sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch clamp was conducted in cultured renal collecting duct M-1 cells to record Na(+) currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na(+) inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ≈2-folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid, which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na(+) retention commonly observed in CHF. © 2015 American Heart Association, Inc.
Urinary proteolytic activation of renal epithelial Na+ channels in chronic heart failure
Zheng, Hong; Liu, Xuefei; Sharma, Neeru M.; Li, Yulong; Pliquett, Rainer U; Patel, Kaushik P.
2015-01-01
One of the key mechanisms involved in renal Na+ retention in chronic heart failure (CHF) is activation of epithelial Na+ channels (ENaC) in collecting tubules. Proteolytic cleavage has an important role in activating ENaC. We hypothesized that enhanced levels of proteases in renal tubular fluid activate ENaC resulting in renal Na+ retention in rats with CHF. CHF was produced by left coronary artery ligation in rats. By immunoblotting, we found that several urinary serine proteases were significantly increased in CHF rats compared to sham rats (fold increases: furin 6.7, prostasin 23.6, plasminogen 2.06 and plasmin 3.57 vs. sham). Similar increases were observed in urinary samples from patients with CHF. Whole-cell patch-clamp was conducted in cultured renal collecting duct M-1 cells to record Na+ currents. Protease-rich urine (from rats and patients with CHF) significantly increased the Na+ inward current in M-1 cells. Two weeks of protease inhibitor treatment significantly abrogated the enhanced diuretic and natriuretic responses to ENaC inhibitor benzamil in rats with CHF. Increased podocyte lesions were observed in the kidneys of rats with CHF by transmission electron microscopy. Consistent with these results, podocyte damage markers desmin and podocin expressions were also increased in rats with CHF (increased ~2 folds). These findings suggest that podocyte damage may lead to increased proteases in the tubular fluid which in turn contributes to the enhanced renal ENaC activity, providing a novel mechanistic insight for Na+ retention commonly observed in CHF. PMID:26628676
Gimenez, Gizeli S.; Coutinho-Neto, Antonio; Kayano, Anderson M.; Simões-Silva, Rodrigo; Trindade, Frances; de Almeida e Silva, Alexandre; Marcussi, Silvana; da Silva, Saulo L.; Fernandes, Carla F. C.; Zuliani, Juliana P.; Calderon, Leonardo A.; Soares, Andreimar M.; Stábeli, Rodrigo G.
2014-01-01
Toxins purified from the venom of spiders have high potential to be studied pharmacologically and biochemically. These biomolecules may have biotechnological and therapeutic applications. This study aimed to evaluate the protein content of Parawixia bistriata venom and functionally characterize its proteins that have potential for biotechnological applications. The crude venom showed no phospholipase, hemorrhagic, or anti-Leishmania activities attesting to low genotoxicity and discrete antifungal activity for C. albicans. However the following activities were observed: anticoagulation, edema, myotoxicity and proteolysis on casein, azo-collagen, and fibrinogen. The chromatographic and electrophoretic profiles of the proteins revealed a predominance of acidic, neutral, and polar proteins, highlighting the presence of proteins with high molecular masses. Five fractions were collected using cation exchange chromatography, with the P4 fraction standing out as that of the highest purity. All fractions showed proteolytic activity. The crude venom and fractions P1, P2, and P3 showed larvicidal effects on A. aegypti. Fraction P4 showed the presence of a possible metalloprotease (60 kDa) that has high proteolytic activity on azo-collagen and was inhibited by EDTA. The results presented in this study demonstrate the presence of proteins in the venom of P. bistriata with potential for biotechnological applications. PMID:24895632
Screening for Proteolytic Activities in Snake Venom by Means of a Multiplexing ESI-MS Assay Scheme
Liesener, A.; Perchuc, Anna-Maria; Schöni, Reto; Wilmer, Marianne; Karst, U.
2005-01-01
A multiplexed mass spectrometry based assay scheme for the simultaneous determination of five different substrate/product pairs was developed as a tool for screening of proteolytic activities in snake venom fractions from Bothrops moojeni. The assay scheme was employed in the functional
Directory of Open Access Journals (Sweden)
Gordon Richard
2012-04-01
Full Text Available Abstract Background The mechanisms of progressive dopaminergic neuronal loss in Parkinson’s disease (PD remain poorly understood, largely due to the complex etiology and multifactorial nature of disease pathogenesis. Several lines of evidence from human studies and experimental models over the last decade have identified neuroinflammation as a potential pathophysiological mechanism contributing to disease progression. Tumor necrosis factor α (TNF has recently emerged as the primary neuroinflammatory mediator that can elicit dopaminergic cell death in PD. However, the signaling pathways by which TNF mediates dopaminergic cell death have not been completely elucidated. Methods In this study we used a dopaminergic neuronal cell model and recombinant TNF to characterize intracellular signaling pathways activated during TNF-induced dopaminergic neurotoxicity. Etanercept and neutralizing antibodies to tumor necrosis factor receptor 1 (TNFR1 were used to block TNF signaling. We confirmed the results from our mechanistic studies in primary embryonic mesencephalic cultures and in vivo using the stereotaxic lipopolysaccharide (LPS model of nigral dopaminergic degeneration. Results TNF signaling in dopaminergic neuronal cells triggered the activation of protein kinase Cδ (PKCδ, an isoform of the novel PKC family, by caspase-3 and caspase-8 dependent proteolytic cleavage. Both TNFR1 neutralizing antibodies and the soluble TNF receptor Etanercept blocked TNF-induced PKCδ proteolytic activation. Proteolytic activation of PKCδ was accompanied by translocation of the kinase to the nucleus. Notably, inhibition of PKCδ signaling by small interfering (siRNA or overexpression of a PKCδ cleavage-resistant mutant protected against TNF-induced dopaminergic neuronal cell death. Further, primary dopaminergic neurons obtained from PKCδ knockout (−/− mice were resistant to TNF toxicity. The proteolytic activation of PKCδ in the mouse substantia nigra in the
Yusmarini1,2)*; R. Indrati1); T. Utami1); Y. Marsono1)
2010-01-01
Some lactic acid bacteria (LAB) strains had been isolated from spontaneously fermented soymilk which have proteolytic system. The purpose of this research was to study ability of isolates in fermentation of soymilk. The changes in bacterial growth, pH, titrable acidity, and proteolytic activities during fermentation were examined. Isolates of Lactobacillus plantarum 1 R.1.3.2; L. plantarum 1 R.11.1.2 and L. acidophilus FNCC 0051 (as a control) were capable growing in soymilk. The results indi...
de Lucio, Héctor; Gamo, Ana María; Ruiz-Santaquiteria, Marta; de Castro, Sonia; Sánchez-Murcia, Pedro A; Toro, Miguel A; Gutiérrez, Kilian Jesús; Gago, Federico; Jiménez-Ruiz, Antonio; Camarasa, María-José; Velázquez, Sonsoles
2017-11-10
The objective of the current study was to enhance the proteolytic stability of peptide-based inhibitors that target critical protein-protein interactions at the dimerization interface of Leishmania infantum trypanothione reductase (Li-TryR) using a backbone modification strategy. To achieve this goal we carried out the synthesis, proteolytic stability studies and biological evaluation of a small library of α/β 3 -peptide foldamers of different length (from 9-mers to 13-mers) and different α→β substitution patterns related to prototype linear α-peptides. We show that several 13-residue α/β 3 -peptide foldamers retain inhibitory potency against the enzyme (in both activity and dimerization assays) while they are far less susceptible to proteolytic degradation than an analogous α-peptide. The strong dependence of the binding affinities for Li-TryR on the length of the α,β-peptides is supported by theoretical calculations on conformational ensembles of the resulting complexes. The conjugation of the most proteolytically stable α/β-peptide with oligoarginines results in a molecule with potent activity against L. infantum promastigotes and amastigotes. Copyright © 2017 Elsevier Masson SAS. All rights reserved.
The influence of radiation on bacterial cells and their proteolytic properties
International Nuclear Information System (INIS)
Szulc, M.; Stefaniakowa, A.; Stanczak, B.; Peconek, J.
1980-01-01
The suspension of bacterial cells and their spores were exposed to X rays in the environment with and without protein. The doses of radiation ranged from 1 to 100 Gy and in case of spores of B. subtilis from 50 to 1000 Gy. It was found that irradiation to Proteus vulgaris, Pseudomonas fluorescens and Ps. aeruginosa caused an inconsiderable decrease of proteolytic properties of the generation originated from irradiated bacteria. Irradiation of B. subtilis spores did not influence the proteolytic activity of bacterial cells derived from the exposed spores. The degree of wasting away of bacteria exposed to the same radiation was higher than the rate of proteolytic properties decrease. The presence of protein in the surroundings had no influence on proteolytic characteristics of new generations. (author)
DEFF Research Database (Denmark)
Jepsen, Malene R.; Kløverpris, Søren; Bøtkjær, Jane A.
2016-01-01
STUDY QUESTION: Is the proteolytic activity of pregnancy-associated plasma protein-A (PAPP-A) regulated by the stanniocalcins (STC1 and STC2) during human follicle maturation? SUMMARY ANSWER: The STCs and PAPP-A show similar expression by immunohistochemistry in developing follicles, and regulation...... of PAPP-A proteolytic activity is suggested by the identification of inhibited protein complexes between PAPP-A and STC1 or STC2 in human follicular fluid (FF). WHAT IS KNOWN ALREADY: The insulin-like growth factor (IGF)-regulating proteinase PAPP-A is secreted by the granulosa cells of estrogen...
Role of Proteolytic Enzymes in the Interaction of Phytopathogenic Microorganisms with Plants.
Valueva, T A; Zaichik, B Ts; Kudryavtseva, N N
2016-12-01
Various forms of participation of proteolytic enzymes in pathogenesis and defense in plants are reviewed. Along with extracellular proteinases, phytopathogenic microorganisms produce specific effectors having proteolytic activity and capable of acting on proteins inside plant cells. In turn, for defense against pathogens, plants use both extracellular and intracellular proteinases.
Multiplex profiling of tumor-associated proteolytic activity in serum of colorectal cancer patients.
Yepes, Diego; Costina, Victor; Pilz, Lothar R; Hofheinz, Ralf; Neumaier, Michael; Findeisen, Peter
2014-06-01
The monitoring of tumor-associated protease activity in blood specimens has recently been proposed as new diagnostic tool in cancer research. In this paper, we describe the screening of a peptide library for identification of reporter peptides (RPs) that are selectively cleaved in serum specimens from colorectal cancer patients and investigate the benefits of RP multiplexing. A library of 144 RPs was constructed that contained amino acid sequences of abundant plasma proteins. Proteolytic cleavage of RPs was monitored with MS. Five RPs that were selectively cleaved in serum specimens from tumor patients were selected for further validation in serum specimens of colorectal tumor patients (n = 30) and nonmalignant controls (n = 60). RP spiking and subsequent quantification of proteolytic fragments with LC-MS showed good reproducibility with CVs always below 26%. The linear discriminant analysis and PCA revealed that a combination of RPs for diagnostic classification is superior to single markers. Classification accuracy reached 88% (79/90) when all five markers were combined. Functional protease profiling with RPs might improve the laboratory-based diagnosis, monitoring and prognosis of malignant disease, and has to be evaluated thoroughly in future studies. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Multiplexed homogeneous assays of proteolytic activity using a smartphone and quantum dots.
Petryayeva, Eleonora; Algar, W Russ
2014-03-18
Semiconductor quantum dot (QD) bioconjugates, with their unique and highly advantageous physicochemical and optical properties, have been extensively utilized as probes for bioanalysis and continue to generate widespread interest for these applications. An important consideration for expanding the utility of QDs and making their use routine is to make assays with QDs more accessible for laboratories that do not specialize in nanomaterials. Here, we show that digital color imaging of QD photoluminescence (PL) with a smartphone camera is a viable, easily accessible readout platform for quantitative, multiplexed, and real-time bioanalyses. Red-, green-, and blue-emitting CdSeS/ZnS QDs were conjugated with peptides that were labeled with a deep-red fluorescent dye, Alexa Fluor 647, and the dark quenchers, QSY9 and QSY35, respectively, to generate Förster resonance energy transfer (FRET) pairs sensitive to proteolytic activity. Changes in QD PL caused by the activity of picomolar to nanomolar concentrations of protease were detected as changes in the red-green-blue (RGB) channel intensities in digital color images. Importantly, measurements of replicate samples made with smartphone imaging and a sophisticated fluorescence plate reader yielded the same quantitative results, including initial proteolytic rates and specificity constants. Homogeneous two-plex and three-plex assays for the activity of trypsin, chymotrypsin, and enterokinase were demonstrated with RGB imaging. Given the ubiquity of smartphones, this work largely removes any instrumental impediments to the adoption of QDs as routine tools for bioanalysis in research laboratories and is a critical step toward the use of QDs for point-of-care diagnostics. This work also adds to the growing utility of smartphones in analytical methods by enabling multiplexed fluorimetric assays within a single sample volume and across multiple samples in parallel.
Isolation and removal of proteolytic enzymes with magnetic cross-linked erythrocytes
International Nuclear Information System (INIS)
Safarik, I. Ivo; Safarikova, Mirka
2001-01-01
New magnetic adsorbents for batch isolation and removal of various proteolytic enzymes were prepared by glutaraldehyde cross-linking of bovine, porcine and human erythrocytes in the presence of fine magnetic particles. Trypsin, chymotrypsin, alkaline bacterial protease and proteases present in various commercial enzyme preparations were efficiently adsorbed on these adsorbents; on the contrary, proteins without proteolytic activity were not adsorbed
Chalabi, Maryam; Khademi, Fatemeh; Yarani, Reza; Mostafaie, Ali
2014-04-01
Actinidin, a member of the papain-like family of cysteine proteases, is abundant in kiwifruit. To date, a few studies have been provided to investigate the proteolytic activity and substrate specificity of actinidin on native proteins. Herein, the proteolytic activity of actinidin was compared to papain on several different fibrous and globular proteins under neutral, acidic and basic conditions. The digested samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and densitometry to assess the proteolytic effect. Furthermore, the levels of free amino nitrogen (FAN) of the treated samples were determined using the ninhydrin colorimetric method. The findings showed that actinidin has no or limited proteolytic effect on globular proteins such as immunoglobulins including sheep IgG, rabbit IgG, chicken IgY and fish IgM, bovine serum albumin (BSA), lipid transfer protein (LTP), and whey proteins (α-lactalbumin and β-lactoglobulin) compared to papain. In contrast to globular proteins, actinidin could hydrolyze collagen and fibrinogen perfectly at neutral and mild basic pHs. Moreover, this enzyme could digest pure α-casein and major subunits of micellar casein especially in acidic pHs. Taken together, the data indicated that actinidin has narrow substrate specificity with the highest enzymatic activity for the collagen and fibrinogen substrates. The results describe the actinidin as a mild plant protease useful for many special applications such as cell isolation from different tissues and some food industries as a mixture formula with other relevant proteases.
EFFECT OF MARINATION WITH PROTEOLYTIC ENZYMES ON QUALITY OF BEEF MUSCLE
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Daniela Istrati
2012-03-01
Full Text Available During storage and thermal treatment meat suffers a number of biochemical and physical-chemical changes in the substrate protein, changes that take place with varying intensity depending on the method of preservation utilized and temperature of thermal treatment applied. Application of different treatments aimed to influence the proteolytic activity as is the case of enzymatic tenderization of beef.Improving the meat tenderness with proteolytic enzymes is promising, but current legislation restricting the use of proteolytic enzymes from bacterial origin and recommended tenderizers salts containing papain, ficin and bromelain. Recent research revealed that meat marinating before grilling results in a reduction of heterocyclic amine content after thermal treatment. Also, the addition of fruit pulp, garlic or other spices contributes to decreased production of heterocyclic amines because of their antioxidant activity. In the present study was aimed influence of exogenous proteolytic enzymes on adult beef tenderness. To increase the tenderness of adult beef were used exogenous enzymes preparations (papain and bromelain and natural sources of enzymes using pineapple and papaya fruit. It was intended to establish the correlation between enzymatic activity of enzymes used in the study, the processing technology and changes in the physical-chemical and biochemical characteristics that occur during storage in refrigerated conditions (evolution of the rigidity index and water holding capacity, cooking losses and cooking yield of the samples injected/marinated with enzymes.
Directory of Open Access Journals (Sweden)
Monica Salamone
Full Text Available In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4 and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs or Serine Integral Membrane Peptidases (SIMPs caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.
Salamone, Monica; Carfì Pavia, Francesco; Ghersi, Giulio
2016-01-01
In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a "resting" phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process.
Salamone, Monica; Carfì Pavia, Francesco
2016-01-01
In vitro cultured endothelial cells forming a continuous monolayer establish stable cell-cell contacts and acquire a “resting” phenotype; on the other hand, when growing in sparse conditions these cells acquire a migratory phenotype and invade the empty area of the culture. Culturing cells in different conditions, we compared expression and clustering of proteolytic enzymes in cells having migratory versus stationary behavior. In order to observe resting and migrating cells in the same microscopic field, a continuous cell monolayer was wounded. Increased expression of proteolytic enzymes was evident in cell membranes of migrating cells especially at sprouting sites and in shed membrane vesicles. Gelatin zymography and western blotting analyses confirmed that in migrating cells, expression of membrane-bound and of vesicle-associated proteolytic enzymes are increased. The enzymes concerned include MMP-2, MMP-9, MT1-MMP, seprase, DPP4 (DiPeptidyl Peptidase 4) and uPA. Shed membrane vesicles were shown to exert degradative activity on ECM components and produce substrates facilitating cell migration. Vesicles shed by migrating cells degraded ECM components at an increased rate; as a result their effect on cell migration was amplified. Inhibiting either Matrix Metallo Proteases (MMPs) or Serine Integral Membrane Peptidases (SIMPs) caused a decrease in the stimulatory effect of vesicles, inhibiting the spontaneous migratory activity of cells; a similar result was also obtained when a monoclonal antibody acting on DPP4 was tested. We conclude that proteolytic enzymes have a synergistic stimulatory effect on cell migration and that their clustering probably facilitates the proteolytic activation cascades needed to produce maximal degradative activity on cell substrates during the angiogenic process. PMID:27152413
Gandhi, Akanksha; Shah, Nagendra P
2014-12-01
The present investigation examined the effects of supplementation of milk peptide fractions produced by enzymatic hydrolysis on the fermentation of reconstituted skim milk (RSM). Changes in pH, cell growth, proteolytic activity, and angiotensin-converting enzyme (ACE)-inhibitory activity were monitored during fermentation of RSM by pure cultures of Lactobacillus acidophilus, Lactobacillus helveticus, Lactobacillus delbrueckii ssp. bulgaricus, and Streptococcus thermophilus. The study showed that supplementation with peptide fractions of different molecular weights did not significantly affect the bacterial growth in RSM. All bacteria showed an increased proteolytic activity in RSM supplemented with large peptides (>10 kDa), and L. helveticus in general exhibited the highest proteolytic activity among the bacteria studied. The ACE-inhibitory activity was observed to be the maximum in RSM supplemented with larger peptides (>10 kDa) for all bacteria. The results suggest that proteolysis by bacteria leads to increased production of ACE-inhibitory peptides compared to the supplemented peptides produced by enzymatic hydrolysis.
Studies on the membrane-associated proteolytic activities of Escherichia coli
International Nuclear Information System (INIS)
Palmer, S.M.
1986-01-01
The membrane fraction contains three proteolytic activities which can be resolved from whole membrane detergent extracts by DEAE-cellulose chromatography. The first two eluting activities have been previously reported as protease V and protease IV. These two enzymes were further purified by gel permeation HPLC. Protease V has a M. W. of 31,000 in SDS-PAGE gels. Protease IV has a M. W. of 62,000 and exists in two distinct isoforms of pl ≅ 6.7 and 6.9. The third enzyme eluting from the DEAE-cellulose column was further purified by affinity chromatography on Benzamidine-Sepharose 6B. This enzyme, referred to herein as protease VI, is a 43 kdal protein which has not been previously characterized. Protease VI was sensitive to inhibition by the serine protease inhibitors phenylmethylsulfony fluoride (PMSF), diisopropylfluorophosphate (DFP) and p-amino-benzamidine (PAB). Additionally, this enzyme exhibited maximal activity at pH ≅ 8.0. Incubation of whole membrane preparations with [ 3 H] DFP resulted in 11 specific proteins acquiring the radioactive label, included in this group of proteins were proteases IV, V, and VI. Several of the DFP-reactive proteins were also shown to bind [ 125 I] ampicillin
Inhibition of proteolytic ferments by taurin in radiation sickness
International Nuclear Information System (INIS)
Dokshina, G.A.; Klimova, A.D.; Yartsev, E.I.; Yakovlev, V.G.
1975-01-01
The application of taurin potassium phosphate (TKPh) as radioprotective remedy resulted in a considerable influence on the activity of the proteolytic ferments in the irradiated organism. White male rats with a weight of 160 to 180 g being kept in cages without any food for 12 hours before this experiment were irradiated with a gamma unit GUM-Co-60 with a dose of 700 rad (LDsub(70/30)). A 4% solution of the preparation was injected in an amount of 40 mg per rat 1, 3, 5, 7 days after irradiation. Under the effect of ionizing radiation there was a progredient increase of proteolytic ferment activity of liver and spleen which was detected already 30 min after irradiation with maximum rate of proteolysis on the 21st day. After injection of the preparation a two-phase reaction developed: on the 7th to 12th day an increased activity of cathepsins in the tissue and in the following time up to the 30th day of observation an inhibition of ferment activity was demonstrated. Simultaneously it was found that the radiation-induced corticosteroid level was prevented by the preparation. A similar effect was also shown by TKPh inhibiting proteolytic ferment activity in experiments with rats with preceding application of hydrocortisone in high doses. The obtained results permit the assumption that the radioprotective activity of taurin comes about by its effect in the direction of structural integrity of the cell membranes leading to a normalization of the hormonal and fermentative events
Elastinolytic and proteolytic enzymes.
Kessler, Efrat; Safrin, Mary
2014-01-01
Pseudomonas aeruginosa secretes into its environment at least seven extracellular proteases: pseudolysin (LasB protease; elastase), aeruginolysin (alkaline proteinase), staphylolysin (staphylolytic endopeptidase; LasA protease), lysyl endopeptidase (protease IV; PrpL), PASP (P. aeruginosa small protease), LepA (Large ExoProtease A), and an aminopeptidase. Their action on host proteins, both individually and synergistically, plays important roles in pathogenesis of P. aeruginosa infections. Methods to measure/detect their activities are fundamental for understanding their physiological functions, roles in pathogenesis, mechanisms of action, regulation, and secretion. Most assays for determination/detection of proteolytic activity employ modified/non-modified casein or gelatin as substrates. In the quantitative assay, fragments generated from azocasein are separated from undigested substrate by trichloroacetic acid precipitation and their absorbance is measured. In non-quantitative assays, proteolytic activity is detected as clearing zones around bacterial growth or samples of culture supernatants on casein containing solid media formed due to local casein degradation. In zymography, individual proteases are detected as clear bands in gelatin/casein containing gels after SDS-PAGE separation, renaturation and protein staining. The elastinolytic capacity of P. aeruginosa is reflected by clearing zones on nutrient agar plates containing insoluble elastin instead of casein. Mueller-Hinton agar plates on which S. aureus cells are grown as a lawn are used to assess the susceptibility of S. aureus isolates to staphylolysin. A clear zone around a staphylolysin-containing sample indicates inhibition of S. aureus growth. Methods for measuring the activity of individual proteases are based on their cleavage specificity. These include assays of elastinolytic activity of pseudolysin and/or staphylolysin using elastin-Congo red as a substrate, a method for determination of
Proteolytic enzymes of lactic acid bacteria
Law, J; Haandrikman, A
The proteolytic system of lactic acid bacteria is essential for their growth in milk and contributes significantly to flavour development in fermented milk products where these microorganisms are used as starter cultures. The proteolytic system is composed of proteinases which initially cleave the
Sirtuins and Proteolytic Systems: Implications for Pathogenesis of Synucleinopathies
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Belém Sampaio-Marques
2015-05-01
Full Text Available Insoluble and fibrillar forms of α-synuclein are the major components of Lewy bodies, a hallmark of several sporadic and inherited neurodegenerative diseases known as synucleinopathies. α-Synuclein is a natural unfolded and aggregation-prone protein that can be degraded by the ubiquitin-proteasomal system and the lysosomal degradation pathways. α-Synuclein is a target of the main cellular proteolytic systems, but it is also able to alter their function further, contributing to the progression of neurodegeneration. Aging, a major risk for synucleinopathies, is associated with a decrease activity of the proteolytic systems, further aggravating this toxic looping cycle. Here, the current literature on the basic aspects of the routes for α-synuclein clearance, as well as the consequences of the proteolytic systems collapse, will be discussed. Finally, particular focus will be given to the sirtuins’s role on proteostasis regulation, since their modulation emerged as a promising therapeutic strategy to rescue cells from α-synuclein toxicity. The controversial reports on the potential role of sirtuins in the degradation of α-synuclein will be discussed. Connection between sirtuins and proteolytic systems is definitely worth of further studies to increase the knowledge that will allow its proper exploration as new avenue to fight synucleinopathies.
Development of a solid-phase assay for measurement of proteolytic enzyme activity
International Nuclear Information System (INIS)
Varani, J.; Johnson, K.; Kaplan, J.
1980-01-01
A solid-phase, plate assay was developed for the measurement of proteolytic enzyme activity. In this assay procedure, radiolabeled substrates were dried onto the surface of microtiter wells. Following drying, the wells were washed two times with saline to remove the nonadherent substrate. When proteolytic enzymes were added to the wells, protein hydrolysis occurred, releasing radioactivity into the supernatant fluid. The amount of protein hydrolysis that occurred was reflected by the amount of radioactivity in the supernatant fluid. When 125 I-hemoglobin was used as the substrate, it was as susceptible to hydrolysis by trypsin in the solid-phase assay as it was in solution in a standard assay procedure. Protease activity from a variety of sources (including from viable cells as well as from extracellular sources) were also able to hydrolyze the hemoglobin on the plate. 125 I-Labeled serum albumen, fibrinogen, and rat pulmonary basement membrane were also susceptible to hydrolysis by trypsin in the solid phase. When [ 14 C]elastin was dried onto the plate, it behaved in a similar manner to elastin in solution. It was resistant to hydrolysis by nonspecific proteases such as trypsin and chymotrypsin but was highly susceptible to hydrolysis by elastase. The solid-phase plate assay has several features which recommended it for routine use. It is as sensitive as standard tube assays (and much more sensitive than routinely used colormetric assays). It is quick and convenient; there are no precipitation, centrifugation, or filtration steps. In addition, very small volumes of radioactive wastes are generated. Another advantage of the solid-phase plate assay is the resistance of the dried substrates to spontaneous breakdown and to microbial contamination. Finally, this assay is suitable for use with viable cells as well as for extracellular proteases
Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis).
Samac, D; Storey, R
1981-12-01
Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling.Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination.
Rivers, David B; Acca, Gillian; Fink, Marc; Brogan, Rebecca; Schoeffield, Andrew
2014-08-01
The spatial distribution of proteolytic enzymes in the adult foregut of Protophormia terraenovae was studied in the context of protein digestion and regurgitation. Based on substrate specificity, pH optima, and use of specific protease inhibitors, all adults tested displayed enzyme activity in the foregut consistent with pepsin, trypsin and chymotrypsin. Chymotrypsin-like and trypsin-like enzyme activity were detected in all gut fluids and tissues tested, with chymotrypsin displaying the highest activity in saliva and salivary gland tissue, whereas maximal trypsin activity was evident in the crop. Pepsin-like activity was only evident in crop fluids and tissues. The activity of all three enzymes was low or undetectable (pepsin) in the fluids and tissue homogenates derived from the esophagus and cardia of any of the adults assayed. Fed adult females displayed higher enzyme activities than fed males, and the activity of all three enzymes were much more prevalent in fed adults than starved. The pH optimum of the trypsin-like enzyme was between pH 7.0 and 8.0; chymotrypsin was near pH 8.0; and maximal pepsin-like activity occurred between pH 1.0 and 2.0. Regurgitate from fed adult females displayed enzyme activity consistent with the proteolytic enzymes detected in crop gut fluids. Enzymes in regurgitate were not derived from food sources based on assays of bovine liver samples. These latter observations suggest that adult flies release fluids from foregut when encountering dry foods, potentially as a means to initiate extra-oral digestion. Copyright © 2014 Elsevier Ltd. All rights reserved.
The proteolytic system of Lactococcus lactis
Kunji, Edmundus Richardus Stephanus
1997-01-01
The bacterium Lactococcus lactis usues an extencive proteolytic system to utilize milk proteins (caseins) in orde to meet its need for amino acids. The genetic and biochemical properties of the putative components of the proteolytic pathway are well-described. However, little is known about the role
Franco, Octávio L; dos Santos, Roseane C; Batista, João A N; Mendes, Ana Cristina M; de Araújo, Marcus Aurélio M; Monnerat, Rose G; Grossi-de-Sá, Maria Fátima; de Freitas, Sonia M
2003-06-01
The cotton boll weevil Anthonomus grandis (Boheman) is one of the major pests of cotton (Gossypium hirsutum L.) in tropical and sub-tropical areas of the New World. This feeds on cotton floral fruits and buds causing severe crop losses. Digestion in the boll weevil is facilitated by high levels of serine proteinases, which are responsible for the almost all proteolytic activity. Aiming to reduce the proteolytic activity, the inhibitory effects of black-eyed pea trypsin/chymotrypsin inhibitor (BTCI), towards trypsin and chymotrypsin from bovine pancreas and from midguts of A. grandis larvae and adult insects were analyzed. BTCI, purified from Vigna unguiculata (L.) seeds, was highly active against different trypsin-like proteinases studied and moderately active against the digestive chymotrypsin of adult insects. Nevertheless, no inhibitory activity was observed against chymotrypsin from A. grandis larval guts. To test the BTCI efficiency in vivo, neonate larvae were reared on artificial diet containing BTCI at 10, 50 and 100 microM. A reduction of larval weight of up to approximately 54% at the highest BTCI concentration was observed. At this concentration, the insect mortality was 65%. This work constitutes the first observation of a Bowman-Birk type inhibitor active in vitro and in vivo toward the cotton boll weevil A. grandis. The results of bioassays strongly suggest that BTCI may have potential as a transgene protein for use in engineered crop plants modified for heightened resistance to the cotton boll weevil.
Imaging proteolytic activity in live cells and animal models.
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Stefanie Galbán
Full Text Available In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors; their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50-100 fold and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspase's was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.
Proteolytic activation transforms heparin cofactor II into a host defense molecule.
Kalle, Martina; Papareddy, Praveen; Kasetty, Gopinath; Tollefsen, Douglas M; Malmsten, Martin; Mörgelin, Matthias; Schmidtchen, Artur
2013-06-15
The abundant serine proteinase inhibitor heparin cofactor II (HCII) has been proposed to inhibit extravascular thrombin. However, the exact physiological role of this plasma protein remains enigmatic. In this study, we demonstrate a previously unknown role for HCII in host defense. Proteolytic cleavage of the molecule induced a conformational change, thereby inducing endotoxin-binding and antimicrobial properties. Analyses employing representative peptide epitopes mapped these effects to helices A and D. Mice deficient in HCII showed increased susceptibility to invasive infection by Pseudomonas aeruginosa, along with a significantly increased cytokine response. Correspondingly, decreased levels of HCII were observed in wild-type animals challenged with bacteria or endotoxin. In humans, proteolytically cleaved HCII forms were detected during wounding and in association with bacteria. Thus, the protease-induced uncovering of cryptic epitopes in HCII, which transforms the molecule into a host defense factor, represents a previously unknown regulatory mechanism in HCII biology and innate immunity.
Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins.
Dallas, David C; Citerne, Florine; Tian, Tian; Silva, Vitor L M; Kalanetra, Karen M; Frese, Steven A; Robinson, Randall C; Mills, David A; Barile, Daniela
2016-04-15
The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. Copyright © 2015 Elsevier Ltd. All rights reserved.
Peptidomic analysis reveals proteolytic activity of kefir microorganisms on bovine milk proteins
Dallas, David C.; Citerne, Florine; Tian, Tian; Silva, Vitor L. M.; Kalanetra, Karen M.; Frese, Steven A.; Robinson, Randall C.; Mills, David A.; Barile, Daniela
2015-01-01
Scope The microorganisms that make up kefir grains are well known for lactose fermentation, but the extent to which they hydrolyze and consume milk proteins remains poorly understood. Peptidomics technologies were used to examine the proteolytic activity of kefir grains on bovine milk proteins. Methods and results Gel electrophoresis revealed substantial digestion of milk proteins by kefir grains, with mass spectrometric analysis showing the release of 609 protein fragments and alteration of the abundance of >1,500 peptides that derived from 27 milk proteins. Kefir contained 25 peptides identified from the literature as having biological activity, including those with antihypertensive, antimicrobial, immunomodulatory, opioid and anti-oxidative functions. 16S rRNA and shotgun metagenomic sequencing identified the principle taxa in the culture as Lactobacillus species. Conclusion The model kefir sample contained thousands of protein fragments released in part by kefir microorganisms and in part by native milk proteases. PMID:26616950
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José Antonio Vázquez
2009-12-01
Full Text Available The aim of this work was to study the recovery of proteolytic and collagenolytic activities from rayfish (Raja clavata viscera wastes. Initially, different parts of the gastrointestinal tract by-products (stomach, duodenum section including pancreas, final intestine were evaluated. The extracts from proximal intestine yielded the highest values of both enzymatic activities. Optimal conditions for protease activity quantification were established at pH = 6, T = 40 °C and incubation time ≤20 min. The mathematical equation used to model the joint effect of pH and temperature led to maximum activity at pH = 8.66 and 59.4 °C, respectively. Overcooled acetone was found to be best option for recovery of enzymatic activities in comparison with ethanol, PEG-4000, ammonium sulphate and ultrafiltration system. Finally, a simple and systematic protocol of partial purification and total recovery of proteases and collagenases was defined.
Cárdenas-Guerra, Rosa Elena; Figueroa-Angulo, Elisa Elvira; Puente-Rivera, Jonathan; Zamudio-Prieto, Olga; Ortega-López, Jaime
2015-01-01
We focus on the iron response of Trichomonas vaginalis to gene family products such as the cysteine proteinases (CPs) involved in virulence properties. In particular, we examined the effect of iron on the gene expression regulation and function of cathepsin L-like and asparaginyl endopeptidase-like CPs as virulence factors. We addressed some important aspects about CPs genomic organization and we offer possible explanations to the fact that only few members of this large gene family are expressed at the RNA and protein levels and the way to control their proteolytic activity. We also summarized all known iron regulations of CPs at transcriptional, posttranscriptional, and posttranslational levels along with new insights into the possible epigenetic and miRNA processes. PMID:26090464
Czech Academy of Sciences Publication Activity Database
Vinterová, Zuzana; Šanda, Miloslav; Dostál, Jiří; Hrušková-Heidingsfeldová, Olga; Pichová, Iva
2011-01-01
Roč. 20, č. 12 (2011), s. 2004-2012 ISSN 0961-8368 R&D Projects: GA MŠk(CZ) LC531; GA ČR GA310/09/1945 Institutional research plan: CEZ:AV0Z40550506 Keywords : Candida parapsilosis * secreted aspartic proteinases * Sapp1p * cell wall * biotin * proteolytic activity Subject RIV: CE - Biochemistry Impact factor: 2.798, year: 2011
Proteolytic and Trypsin Inhibitor Activity in Germinating Jojoba Seeds (Simmondsia chinensis) 1
Samac, Deborah; Storey, Richard
1981-01-01
Changes in proteolytic activity (aminopeptidase, carboxypeptidase, endopeptidase) were followed during germination (imbibition through seedling development) in extracts from cotyledons of jojoba seeds (Simmondsia chinensis). After imbibition, the cotyledons contained high levels of sulfhydryl aminopeptidase activity (APA) but low levels of serine carboxypeptidase activity (CPA). CPA increased with germination through the apparent loss of a CPA inhibitor substance in the seed. Curves showing changes in endopeptidase activity (EPA) assayed at pH 4, 5, 6, 7, and 8 during germination were distinctly different. EPA at pH 4, 5, 6, and 7 showed characteristics of sulfhydryl enzymes while activity at pH 8 was probably due to a serine type enzyme. EPA at pH 6 was inhibited early in germination by one or more substances in the seed. Activities at pH 5 and later at pH 6 were the highest of all EPA throughout germination and increases in these activities were associated with a rapid loss of protein from the cotyledons of the developing seedling. Jojoba cotyledonary extracts were found to inhibit the enzymic activity of trypsin, chymotrypsin, and pepsin but not the protease from Aspergillus saotoi. The heat-labile trypsin inhibitor substance(s) was found in commercially processed jojoba seed meal and the albumin fraction of seed proteins. Trypsin inhibitor activity decreased with germination. PMID:16662104
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Gastón Ezequiel Ortiz
2016-01-01
Full Text Available A comparative study of the proteolytic enzymes production using twelve Aspergillus strains previously unused for this purpose was performed by solid state fermentation. A semiquantitative and quantitative evaluation of proteolytic activity were carried out using crude enzymatic extracts obtained from the fermentation cultures, finding seven strains with high and intermediate level of protease activity. Biochemical, thermodynamics, and kinetics features such as optimum pH and temperature values, thermal stability, activation energy (Ea, quotient energy (Q10, Km, and Vmax were studied in four enzymatic extracts from the selected strains that showed the highest productivity. Additionally, these strains were evaluated by zymogram analysis obtaining protease profiles with a wide range of molecular weight for each sample. From these four strains with the highest productivity, the proteolytic extract of A. sojae ATCC 20235 was shown to be an appropriate biocatalyst for hydrolysis of casein and gelatin substrates, increasing its antioxidant activities in 35% and 125%, respectively.
Shori, A B; Baba, A S; Keow, J N
2012-12-15
There is an increasing demand of functional foods in developed countries. Yogurt plays an important role in the management of blood pressure. Several bioactive peptides isolated from Allium sativum or fish collagen have shown antihypertensive activity. Thus, in the present study the effects of A. sativum and/or Fish Collagen (FC) on proteolysis and ACE inhibitory activity in yogurt (0, 7 and 14 day) and cheese (0, 14 and 28 day) were investigated. Proteolytic activities were the highest on day 7 of refrigerated storage in A. sativum-FC-yogurt (337.0 +/- 5.3 microg g(-1)) followed by FC-yogurt (275.3 +/- 2.0 microg g(-1)), A. sativum-yogurt (245.8 +/- 4.2 microg g(-1)) and plain-yogurt (40.4 +/- 1.2 microg g(-1)). On the other hand, proteolytic activities in cheese ripening were the highest (p sativum-cheeses (411.4 +/- 4.3 and 528.7 +/- 1.6 microg g(-1), respectively). However, the presence of FC increased the proteolysis to the highest level on day 28 of storage for FC- and A. sativum-FC cheeses (641.2 +/- 0.1 and 1128.4 +/- 4.5 microg g(-1), respectively). In addition, plain- and A. sativum-yogurts with or without FC showed maximal inhibition of ACE on day 7 of storage. Fresh plain- and A. sativum-cheeses showed ACE inhibition (72.3 +/- 7.8 and 50.4 +/- 1.6 % respectively), the presence of FC in both type of cheeses reduced the ACE inhibition to 62.9 +/- 0.8 and 44.5 +/- 5.0%, respectively. However, refrigerated storage increased ACE inhibition in cheeses (p sativum-yogurt or cheese enhanced the proteolytic activity. Thus, it has potential in the development of an effective dietary strategy for hypertension associated cardiovascular diseases.
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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.
Matagne, André; Bolle, Laetitia; El Mahyaoui, Rachida; Baeyens-Volant, Danielle; Azarkan, Mohamed
2017-06-01
Crude pineapple proteases extract (aka stem bromelain; EC 3.4.22.4) is an important proteolytic mixture that contains enzymes belonging to the cysteine proteases of the papain family. Numerous studies have been reported aiming at the fractionation and characterization of the many molecular species present in the extract, but more efforts are still required to obtain sufficient quantities of the various purified protease forms for detailed physicochemical, enzymatic and structural characterization. In this work, we describe an efficient strategy towards the purification of at least eight enzymatic forms. Thus, following rapid fractionation on a SP-Sepharose FF column, two sub-populations with proteolytic activity were obtained: the unbound (termed acidic) and bound (termed basic) bromelain fractions. Following reversible modification with monomethoxypolyethylene glycol (mPEG), both fractions were further separated on Q-Sepharose FF and SP-Sepharose FF, respectively. This procedure yielded highly purified molecular species, all titrating ca. 1 mol of thiol group per mole of enzyme, with distinct biochemical properties. N-terminal sequencing allowed identifying at least eight forms with proteolytic activity. The basic fraction contained previously identified species, i.e. basic bromelain forms 1 and 2, ananain forms 1 and 2, and comosain (MEROPS identifier: C01.027). Furthermore, a new proteolytic species, showing similarities with basic bomelain forms 1 and 2, was discovered and termed bromelain form 3. The two remaining species were found in the acidic bromelain fraction and were arbitrarily named acidic bromelain forms 1 and 2. Both, acidic bromelain forms 1, 2 and basic bromelain forms 1, 2 and 3 are glycosylated, while ananain forms 1 and 2, and comosain are not. The eight protease forms display different amidase activities against the various substrates tested, namely small synthetic chromogenic compounds (DL-BAPNA and Boc-Ala-Ala-Gly-pNA), fluorogenic compounds
Angiogenic activity of bFGF and VEGF suppressed by proteolytic cleavage by neutrophil elastase
International Nuclear Information System (INIS)
Ai, Shingo; Cheng Xianwu; Inoue, Aiko; Nakamura, Kae; Okumura, Kenji; Iguchi, Akihisa; Murohara, Toyoaki; Kuzuya, Masafumi
2007-01-01
Neutrophil elastase (NE), a serine protease released from the azurophil granules of activated neutrophil, proteolytically cleaves multiple cytokines, and cell surface proteins. In the present study, we examined whether NE affects the biological abilities of angiogenic growth factors such as basic-fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). NE degraded bFGF and VEGF in a time- and concentration-dependent manner, and these degradations were suppressed by sivelestat, a synthetic inhibitor of NE. The bFGF- or VEGF-mediated proliferative activity of human umbilical vein endothelial cells was inhibited by NE, and the activity was recovered by sivelestat. Furthermore, NE reduced the bFGF- or VEGF-induced tubulogenic response of the mice aortas, ex vivo angiogenesis assay, and these effects were also recovered by sivelestat. Neutrophil-derived NE degraded potent angiogenic factors, resulting in loss of their angiogenic activity. These findings provide additional insight into the role played by neutrophils in the angiogenesis process at sites of inflammation
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L. V. Antipova
2016-01-01
Full Text Available Enzymatic technologies were included strongly into practical activities of the person, the volume of the world market constantly grows and is updated. However the domestic production of enzymatic preparations very lags behind world level that is in many respects connected with insufficient scientific and technical base for a wide circulation of technologies in large-scale production. At the same time there were Russian producers of enzymatic preparations from animal fabrics and bodies for processing of raw materials of an animal origin, according to forecasts, of interest in rational use of resources of an animal origin. In article data on research of properties of the enzymatic preparation "Protepsin" and an assessment of prospects of application are provided in processing of raw materials of an animal origin. The enzymatic preparation "Protepsin" made in the conditions of JSC Plant of Endocrine Enzymes (Rzhavki, Moscow region activity at action on proteins of meat shows, including the strengthened structure, has milk-clotting effect, is active in the field of pH 4,0-6,0 and temperature 20-45zs. The proteinaceous complex includes 4 fractions, 2 from which possess the general proteolytic activity. One of them shows the general proteolytic and milk-clotting activity. Enzymes differ in an amino-acid set and molecular weight. The method of a disk electrophoresis determined molecular-mass structure of "Protepsin". The preparation inactivation conditions guaranteeing its safety in the production technology of foodstuff as active proteolytic enzymes in the course of digestion can cause violations of integrity of fabrics and corresponding diseases are shown. Thus, conditions of use of a perspective proteolytic preparation in technology of a wide range of food of an animal origin are in a complex proved and picked up.
Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin
International Nuclear Information System (INIS)
Hill-Eubanks, D.C.; Parker, C.G.; Lollar, P.
1989-01-01
Blood coagulation factor VIII (fVIII) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. The authors isolated a serine protease from Bothrops jararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO 4 /PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated 125 I-fVIII to function as a cofactor in the activation of factor X by factor IXa, they found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by light-chain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function
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Keyvan Pakshir
2016-12-01
Conclusion: This study indicated that hemolysin and proteolytic enzymes potentially play a role in dermatophyte pathogenesis and S. aureus could be considered as a main bacterium for creation of co-hemolytic effect in association with dermatophyte species.
DEFF Research Database (Denmark)
Nissen, Mogens Holst; Claësson, M H
1987-01-01
the endogenous production of interleukin 2 in the MLC culture; monoclonal antibody which reacts with both the native beta 2-m and M-beta 2-m molecule blocks the augmentation of cytotoxic T lymphocyte production induced by M-beta 2-m; murine as well as human MLC responder cells can proteolytically modify native......A proteolytically modified form of beta 2-microglobulin (beta 2-m) present in the serum of patients suffering from autoimmune, immunodeficient diseases and cancer has been reported in the literature. In the present study we show that human beta 2-m as well as the proteolytically modified human form...... (M-beta 2-m) bind to murine lymphocytes expressing H-2 class I antigens; M-beta 2-m, when added at day 0 and 1 of culture in nanomolar concentrations to a one-way murine allogeneic mixed lymphocyte culture (MLC) augments the generation of specific cytotoxic T lymphocytes; M-beta 2-m increases...
The epithelial sodium channel γ-subunit is processed proteolytically in human kidney
DEFF Research Database (Denmark)
Langkilde, Rikke Zachar; Skjødt, Karsten; Marcussen, Niels
2015-01-01
The epithelial sodium channel (ENaC) of the kidney is necessary for extracellular volume homeostasis and normal arterial BP. Activity of ENaC is enhanced by proteolytic cleavage of the gamma-subunit and putative release of a 43-amino acid inhibitory tract from the gamma-subunit ectodomain. We......ENaC was detected consistently only in tissue from patients with proteinuria and observed in collecting ducts. In conclusion, human kidney gammaENaC is subject to proteolytic cleavage, yielding fragments compatible with furin cleavage, and proteinuria is associated with cleavage at the putative prostasin...
Persistence of bacterial proteolytic enzymes in lake ecosystems.
Kiersztyn, Bartosz; Siuda, Waldemar; Chróst, Ryszard J
2012-04-01
This study analyzes proteolytic enzyme persistence and the role of dead (or metabolically inactive) aquatic bacteria in organic matter cycling. Samples from four lakes of different trophic status were used. Irrespective of the trophic status of the examined lakes, bacterial aminopeptidases remained active even 72 h after the death of the bacteria that produced them. The total pool of proteolytic enzymes in natural lake water samples was also stable. We found that the rates of amino acid enzymatic release from proteinaceous matter added to preserved lake water sample were constant for at least 96 h (r(2) = 0.99, n = 17, P ≤ 0.0001, V(max) = 84.6 nM h(-1) ). We also observed that proteases built into bacterial cell debris fragments remained active for a long time, even after the total destruction of cells. Moreover, during 24 h of incubation time, about 20% of these enzymatically active fragments adsorbed onto natural seston particles, becoming a part of the 'attached enzymes system' that is regarded as the 'hot-spot' of protein degradation in aquatic ecosystems. © 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.
Characterisation of a novel proteolytic enzyme localised to goblet cells in rat and man
DEFF Research Database (Denmark)
Poulsen, Steen Seier
1984-01-01
A proteolytic enzyme, ingobsin , purified from rat duodenal extracts is shown to be localised to intestinal goblet cells of both man and rat. Enzyme positive cells decrease in number from duodenum to colon. The enzyme is a 33 000 Mr protein with an isoelectric point of 5.1. The pH optimum...... for enzymatic activity is 7.4-8.0. Based on substrate specificity for arg-x, lys-x and to a lesser degree tyr-x, on the effect of diisopropylphosphorofluoride , Trasylol and phenylmethylsulfonylfluoride and on proteolytic activity towards intact proteins, ingobsin is classified as a serine proteinase...
Research Applications of Proteolytic Enzymes in Molecular Biology
Directory of Open Access Journals (Sweden)
József Tőzsér
2013-11-01
Full Text Available Proteolytic enzymes (also termed peptidases, proteases and proteinases are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications require their use, including production of Klenow fragments, peptide synthesis, digestion of unwanted proteins during nucleic acid purification, cell culturing and tissue dissociation, preparation of recombinant antibody fragments for research, diagnostics and therapy, exploration of the structure-function relationships by structural studies, removal of affinity tags from fusion proteins in recombinant protein techniques, peptide sequencing and proteolytic digestion of proteins in proteomics. The aim of this paper is to review the molecular biological aspects of proteolytic enzymes and summarize their applications in the life sciences.
Lim, Seng Koon; Sandén, Camilla; Selegård, Robert; Liedberg, Bo; Aili, Daniel
2016-02-01
Membrane active peptides are of large interest for development of drug delivery vehicles and therapeutics for treatment of multiple drug resistant infections. Lack of specificity can be detrimental and finding routes to tune specificity and activity of membrane active peptides is vital for improving their therapeutic efficacy and minimize harmful side effects. We describe a de novo designed membrane active peptide that partition into lipid membranes only when specifically and covalently anchored to the membrane, resulting in pore-formation. Dimerization with a complementary peptide efficiently inhibits formation of pores. The effect can be regulated by proteolytic digestion of the inhibitory peptide by the matrix metalloproteinase MMP-7, an enzyme upregulated in many malignant tumors. This system thus provides a precise and specific route for tuning the permeability of lipid membranes and a novel strategy for development of recognition based membrane active peptides and indirect enzymatically controlled release of liposomal cargo.
DEFF Research Database (Denmark)
Skjøt-Arkil, Helene; Barascuk, Natasha; Register, Thomas
2010-01-01
in almost all stages of atherosclerosis, by both initiating atherosclerotic plaques and degrading them through the secretion of proteolytic enzymes leading to rupture. This review summarizes the literature on the role of macrophages and their proteolytic activity on proteins in the extracellular matrix (ECM......) of the atherosclerotic plaque with a view to suggest a novel approach for identification of vulnerable plaques and turnover by the use of a new type of biomarker. The PubMed database was searched using the terms macrophages, foam cells, atherosclerosis, CVD, ECM remodeling, biomarker, neoepitope, matrix...... of the constituents of the ECM of the atherosclerotic plaque. At present it is not clear which proteases play pivotal roles at distinct stages of pathogenesis, rather that the combined proteolytic potential with some proteases at early stages and other at later stages may result in plaque rupture. This macrophage...
Membrane-associated proteolytic activity in Escherichia coli that is stimulated by ATP
International Nuclear Information System (INIS)
Klemes, Y.; Voellmy, R.W.; Goldberg, A.L.
1986-01-01
The degradation of proteins in bacteria requires metabolism energy. One important enzyme in this process is protease La, a soluble ATP-dependent protease encoded by the lon gene. However, lon mutants that lack a functional protease La still show some ATP-dependent protein breakdown. The authors have reported an ATP-stimulated endoproteolytic activity associated with the inner membrane of E. coli. This ATP-stimulated activity is found in normal levels in membranes derived from lon mutants, including strains carrying insertions in the lon gene. The membrane-bound activity hydrolyzes 14 C-methylglobin at a linear rate for up to 3 hours. These fractions also contain appreciable proteolytic activity that is not affected by ATP. The stimulation by ATP requires the presence of Mg 2+ . Nonhydrolyzable ATP analogs (e.g. AMPPNP or ATP-γ-S) and ADP do not enhance proteolysis. Unlike protease La, the membrane-associated enzyme does not degrade the fluorometric substrate, Glt-Ala-Ala-Phe-MNA, in an ATP-stimulated fashion, and its level is not influenced by high temperature of by the gene which regulates the heat-shock response. The enzyme is inhibited by dichloroisocoumarin and certain peptide chloromethyl ketones. They conclude that E. coli contain at least two ATP-dependent proteases with distinct specificities: one is soluble and the other is membrane-associated
Proteolytic signatures define unique thrombin-derived peptides present in human wound fluid in vivo.
Saravanan, Rathi; Adav, Sunil S; Choong, Yeu Khai; van der Plas, Mariena J A; Petrlova, Jitka; Kjellström, Sven; Sze, Siu Kwan; Schmidtchen, Artur
2017-10-13
The disease burden of failing skin repair and non-healing ulcers is extensive. There is an unmet need for new diagnostic approaches to better predict healing activity and wound infection. Uncontrolled and excessive protease activity, of endogenous or bacterial origin, has been described as a major contributor to wound healing impairments. Proteolytic peptide patterns could therefore correlate and "report" healing activity and infection. This work describes a proof of principle delineating a strategy by which peptides from a selected protein, human thrombin, are detected and attributed to proteolytic actions. With a particular focus on thrombin-derived C-terminal peptides (TCP), we show that distinct peptide patterns are generated in vitro by the human S1 peptidases human neutrophil elastase and cathepsin G, and the bacterial M4 peptidases Pseudomonas aeruginosa elastase and Staphylococcus aureus aureolysin, respectively. Corresponding peptide sequences were identified in wound fluids from acute and non-healing ulcers, and notably, one peptide, FYT21 (FYTHVFRLKKWIQKVIDQFGE), was only present in wound fluid from non-healing ulcers colonized by P. aeruginosa and S. aureus. Our result is a proof of principle pointing at the possibility of defining peptide biomarkers reporting distinct proteolytic activities, of potential implication for improved diagnosis of wound healing and infection.
International Nuclear Information System (INIS)
Gertman, V.Z.
1982-01-01
The proteolytic activity of trypsine-like proteases of blood at acute injury period doesn't increase as in animals with mere burn and in animals with nure irradiation as well. It can be explained by the absence of adequate stress reaction of the organism due to the sharp decrease of organism reactivity in case of combined burn and radiation injury. Application of low temperatures and combination of cryogenic effect and RZP-3 preparation promotes proteolytic activity increase in animals with combined burn and radiation injury in the climax of radiation sickness. The normalization of the factor was observed at late periods of the investigation. It can be regarded as recovery of the organism reactivity
Baraldi, Patrícia T; Magro, Angelo J; Matioli, Fábio F; Marcussi, Silvana; Lemke, Ney; Calderon, Leonardo A; Stábeli, Rodrigo G; Soares, Andreimar M; Correa, Arlene G; Fontes, Marcos R M
2016-02-01
Metalloproteases play a fundamental role in snake venom envenomation inducing hemorrhagic, fibrigen(ogen)olytic and myotoxic effects in their victims. Several snake venoms, such as those from the Bothrops genus, present important local effects which are not efficiently neutralized by conventional serum therapy. Consequently, these accidents may result in permanent sequelae and disability, creating economic and social problems, especially in developing countries, leading the attention of the World Health Organization that considered ophidic envenomations a neglected tropical disease. Aiming to produce an efficient inhibitor against bothropic venoms, we synthesized different molecules classified as quinolinones - a group of low-toxic chemical compounds widely used as antibacterial and antimycobacterial drugs - and tested their inhibitory properties against hemorrhage caused by bothropic venoms. The results from this initial screening indicated the molecule 2-hydroxymethyl-6-methoxy-1,4-dihydro-4-quinolinone (Q8) was the most effective antihemorrhagic compound among all of the assayed synthetic quinolinones. Other in vitro and in vivo experiments showed this novel compound was able to inhibit significantly the hemorrhagic and/or proteolytic activities of bothropic crude venoms and isolated snake venom metalloproteases (SVMPs) even at lower concentrations. Docking and molecular dynamic simulations were also performed to get insights into the structural basis of Q8 inhibitory mechanism against proteolytic and hemorrhagic SVMPs. These structural studies demonstrated that Q8 may form a stable complex with SVMPs, impairing the access of substrates to the active sites of these toxins. Therefore, both experimental and structural data indicate that Q8 compound is an interesting candidate for antiophidic therapy, particularly for the treatment of the hemorrhagic and necrotic effects induced by bothropic venoms. Copyright © 2015 Elsevier B.V. and Société Française de
Alen'kina, S A; Nikitina, V E
2015-01-01
The lectins of associative nitrogen-fixing strains Azospirillum brasilense Sp7 and Sp245 were shown to exerte a multidirectional effect on the activity of acidic (pH 3.5), neutral (6.8), and alkaline (pH 7.8) proteinases. The lectin of the epiphytic A. brasilense Sp7 decreased proteolytic activity at all pH values, whereas the lectin of the endophytic A. brasilense Sp245 activated neutral and alkaline proteinases, while not affecting the alkaline ones. Experiments with protease inhibitors made it possible to conclude that the lectins of the studied A. brasilense strains alter the ratio between the activities of different protease types in germinating seeds. The activity of trypsin inhibitors in wheat seedling roots was found to increase in the presence of the lectins. Our results indicate a broader spectrum of effects of azospirilla lectins on the host plant organism.
Dual regulatory switch confers tighter control on HtrA2 proteolytic activity.
Singh, Nitu; D'Souza, Areetha; Cholleti, Anuradha; Sastry, G Madhavi; Bose, Kakoli
2014-05-01
High-temperature requirement protease A2 (HtrA2), a multitasking serine protease that is involved in critical biological functions and pathogenicity, such as apoptosis and cancer, is a potent therapeutic target. It is established that the C-terminal post-synaptic density protein, Drosophila disc large tumor suppressor, zonula occludens-1 protein (PDZ) domain of HtrA2 plays pivotal role in allosteric modulation, substrate binding and activation, as commonly reported in other members of this family. Interestingly, HtrA2 exhibits an additional level of functional modulation through its unique N-terminus, as is evident from 'inhibitor of apoptosis proteins' binding and cleavage. This phenomenon emphasizes multiple activation mechanisms, which so far remain elusive. Using conformational dynamics, binding kinetics and enzymology studies, we addressed this complex behavior with respect to defining its global mode of regulation and activity. Our findings distinctly demonstrate a novel N-terminal ligand-mediated triggering of an allosteric switch essential for transforming HtrA2 to a proteolytically competent state in a PDZ-independent yet synergistic activation process. Dynamic analyses suggested that it occurs through a series of coordinated structural reorganizations at distal regulatory loops (L3, LD, L1), leading to a population shift towards the relaxed conformer. This precise synergistic coordination among different domains might be physiologically relevant to enable tighter control upon HtrA2 activation for fostering its diverse cellular functions. Understanding this complex rheostatic dual switch mechanism offers an opportunity for targeting various disease conditions with tailored site-specific effector molecules. © 2014 FEBS.
DEFF Research Database (Denmark)
Kløverpris, Søren; Mikkelsen, Jakob Hauge; Pedersen, Josefine Hvidkjær
2015-01-01
regulation in these species. Several physiological functions of STC1 have been reported, although many molecular details are still lacking. We here demonstrate that STC1 is an inhibitor of the metzincin metalloproteinase, pregnancy-associated plasma protein-A (PAPP-A), which modulates insulin-like growth...... that the homologous STC2 inhibits PAPP-A proteolytic activity, and that this depends on the formation of a covalent complex between the inhibitor and the proteinase, mediated by Cys-120 of STC2. We find that STC1 is unable to bind covalently to PAPP-A, in agreement with the absence of a corresponding cysteine residue....... It rather binds to PAPP-A with high affinity (KD = 75 pm). We further demonstrate that both STC1 and STC2 show inhibitory activity toward PAPP-A2, but not selected serine proteinases and metalloproteinases. We therefore conclude that the STCs are proteinase inhibitors, probably restricted in specificity...
Choi, Nack-Shick; Choi, Jong Hyun; Kim, Bo-Hye; Han, Yun-Jon; Kim, Joong Su; Lee, Seung-Goo; Song, Jae Jun
2009-06-01
A new zymography method for simultaneous detection of two different enzymatic activities (lipolytic and proteolytic) using a single SDS-containing or native-conformation gel and a mixed-substrate (glycerol tributyrate and fibrin) (MS)(1) gel was developed. After routine electrophoresis, SDS in the gel was removed by treatment with Triton X-100. Gel proteins were electrotransferred to the MS gel. To visualize lipolytic activity, the MS gel was incubated at 37 degrees C (for 6 or 24 h) until clear bands against an opaque background were observed. To detect proteolytic activity, the same MS gel was stained with Coomassie brilliant blue. Using this method, we show that six lipolytic enzymes from Staphylococcus pasteuri NJ-1 and four proteolytic enzymes from two Bacillus strains, B. licheniformis DJ-2 and B. licheniformis NJ-5, isolated from soil, can be simultaneously detected.
MHC molecules protect T cell epitopes against proteolytic destruction
DEFF Research Database (Denmark)
Mouritsen, S; Meldal, M; Werdelin, O
1992-01-01
There is a subtle duality in the role of proteolytic enzymes in Ag processing. They are required to fragment protein Ag ingested by APC. However, prolonged exposure to proteolytic enzymes may lead to a complete degradation of the Ag, leaving nothing for the T cell system to recognize. What ensures...
Papagianni, Maria
2014-01-01
A number of novel Penicillium strains belonging to Penicillium nalgiovense, Penicillium solitum, Penicillium commune, Penicillium olsonii, and Penicillium oxalicum species, isolated from the surface of traditional Greek sausages, were evaluated for their proteolytic and lipolytic potential in a solid substrate first and next in submerged fermentations, using complex media. Extracellular proteolytic activity was assessed at acid, neutral, and alkaline pH, while the lipolytic activity was assessed using olive oil, the short-chain triacylglycerol tributyrin, and the long-chain triolein, as substrates. The study revealed that although closely related, the tested strains produce enzymes of distinct specificities. P. nalgiovense PNA9 produced the highest alkaline proteolytic activity (13.2 unit (U)/ml) and the highest lipolytic activity with tributyrin (92 U/ml). Comparisons with known sources show that proteases and/or lipases can be secreted effectively by some Penicillia (P. nalgiovense PNA4, PNA7, and PNA9 and P. solitum PSO1), and further investigations on their properties and characteristics would be promising.
Research Applications of Proteolytic Enzymes in Molecular Biology
Mótyán, János András; Tóth, Ferenc; Tőzsér, József
2013-01-01
Proteolytic enzymes (also termed peptidases, proteases and proteinases) are capable of hydrolyzing peptide bonds in proteins. They can be found in all living organisms, from viruses to animals and humans. Proteolytic enzymes have great medical and pharmaceutical importance due to their key role in biological processes and in the life-cycle of many pathogens. Proteases are extensively applied enzymes in several sectors of industry and biotechnology, furthermore, numerous research applications ...
Effect of wine inhibitors on the proteolytic activity of papain from Carica papaya L. latex.
Benucci, Ilaria; Esti, Marco; Liburdi, Katia
2015-01-01
The influence of potential inhibitors naturally present in wine on the proteolytic activity of papain from Carica papaya latex was investigated to evaluate its applicability in white wine protein haze stabilization. Enzymatic activity was tested against a synthetic tripeptide chromogenic substrate in wine-like acidic medium that consisted of tartaric buffer (pH 3.2) supplemented with ethanol, free sulfur dioxide (SO2 ), grape skin and seed tannins within the average ranges of concentrations that are typical in wine. The diagnosis of inhibition type, performed with the graphical method, demonstrated that all of tested wine constituents were reversible inhibitors of papain. The strongest inhibition was exerted by free SO2 , which acted as a mixed-type inhibitor, similar to grape skin and seed tannins. Finally, when tested in table white wines, the catalytic activity of papain, even when if it was ascribable to the hyperbolic behavior of Michaelis-Menten equation, was determined to be strongly affected by free SO2 and total phenol level. © 2014 American Institute of Chemical Engineers.
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
Saperas, Nuria; Fonfria-Subiros, Elsa
2011-01-01
This laboratory exercise uses a problem-based approach to expose students to some basic concepts relating to proteins and enzymes. One of the main applications of enzymes at the industrial level is their use in the detergent market. The students examine a detergent sample to ascertain whether proteolytic enzymes are a component and, if so, which…
Proteolytic stability in colloidal systems : interaction of proteins with the solid-water interface
Maste, M.C.L.
1996-01-01
Proteolytic enzymes in liquid detergents suffer from lack of stability in the sense that activity diminishes with time. Although the phenomenon could be attributed to several factors, the influence of colloidal surfaces on the enzymatic stability was investigated. Besides the types of
Proteolytic crosstalk in multi-protease networks
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.
Han, Peng; Niu, Chang-Ying; Biondi, Antonio; Desneux, Nicolas
2012-11-01
The transgenic Cry1Ac (Bt toxin) + CpTI (Cowpea Trypsin Inhibitor) cotton cultivar CCRI41 is increasingly used in China and potential side effects on the honey bee Apis mellifera L. have been documented recently. Two studies have assessed potential lethal and sublethal effects in young bees fed with CCRI41 cotton pollen but no effect was observed on learning capacities, although lower feeding activity in exposed honey bees was noted (antifeedant effect). The present study aimed at providing further insights into potential side effects of CCRI41 cotton on honey bees. Emerging honey bees were exposed to different pollen diets using no-choice feeding protocols (chronic exposure) in controlled laboratory conditions and we aimed at documenting potential mechanisms underneath the CCRI41 antifeedant effect previously reported. Activity of midgut proteolytic enzyme of young adult honey bees fed on CCRI41 cotton pollen were not significantly affected, i.e. previously observed antifeedant effect was not linked to disturbed activity of the proteolytic enzymes in bees' midgut. Hypopharyngeal gland development was assessed by quantifying total extractable proteins from the glands. Results suggested that CCRI41 cotton pollen carries no risk to hypopharyngeal gland development of young adult honey bees. In the two bioassays, honey bees exposed to 1 % soybean trypsin inhibitor were used as positive controls for both midgut proteolytic enzymes and hypopharyngeal gland proteins quantification, and bees exposed to 48 ppb (part per billion) (i.e. 48 ng g(-1)) imidacloprid were used as controls for exposure to a sublethal concentration of toxic product. The results show that the previously reported antifeedant effect of CCRI41 cotton pollen on honey bees is not linked to effects on their midgut proteolytic enzymes or on the development of their hypopharyngeal glands. The results of the study are discussed in the framework of risk assessment of transgenic crops on honey bees.
Kee, Nalise Low Ah; Krause, Jason; Blatch, Gregory L; Muramoto, Koji; Sakka, Kazuo; Sakka, Makiko; Naudé, Ryno J; Wagner, Leona; Wolf, Raik; Rahfeld, Jens-Ulrich; Demuth, Hans-Ulrich; Mielicki, Wojciech P; Frost, Carminita L
2015-10-01
Proteases are essential for tumour progression and many are over-expressed during this time. The main focus of research was the role of these proteases in degradation of the basement membrane and extracellular matrix (ECM), thereby enabling metastasis to occur. Cancer procoagulant (CP), a protease present in malignant tumours, but not normal tissue, is a known activator of coagulation factor X (FX). The present study investigated the function of CP in cancer progression by focussing on its enzymatic specificity. FX cleavage was confirmed using SDS-PAGE and MALDI-TOF MS and compared to the proteolytic action of CP on ECM proteins, including collagen type IV, laminin and fibronectin. Contrary to previous reports, CP cleaved FX at the conventional activation site (between Arg-52 and Ile-53). Additionally, degradation of FX by CP occurred at a much slower rate than degradation by conventional activators. Complete degradation of the heavy chain of FX was only visible after 24 h, while degradation by RVV was complete after 30 min, supporting postulations that the procoagulant function of CP may be of secondary importance to its role in cancer progression. Of the ECM proteins tested, only fibronectin was cleaved. The substrate specificity of CP was further investigated by screening synthetic peptide substrates using a novel direct CP assay. The results indicate that CP is not essential for either cancer-associated blood coagulation or the degradation of ECM proteins. Rather, they suggest that this protease may be required for the proteolytic activation of membrane receptors.
International Nuclear Information System (INIS)
Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi
2011-01-01
The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 μM) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 μM) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKCδ) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 μM). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKCδ D327A and kinase dead PKCδ K376R or siRNA-mediated knockdown of PKCδ protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKCδ promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKCδ expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKCδ cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKCδ D327A protein protected against 6-OHDA-induced PKCδ activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKCδ is a key downstream event in dopaminergic degeneration, and these results may have important translational value for development of novel treatment strategies for PD.
Simmons, Graham; Zmora, Pawel; Gierer, Stefanie; Heurich, Adeline; Pöhlmann, Stefan
2013-12-01
The severe acute respiratory syndrome (SARS) pandemic revealed that zoonotic transmission of animal coronaviruses (CoV) to humans poses a significant threat to public health and warrants surveillance and the development of countermeasures. The activity of host cell proteases, which cleave and activate the SARS-CoV spike (S) protein, is essential for viral infectivity and constitutes a target for intervention. However, the identities of the proteases involved have been unclear. Pioneer studies identified cathepsins and type II transmembrane serine proteases as cellular activators of SARS-CoV and demonstrated that several emerging viruses might exploit these enzymes to promote their spread. Here, we will review the proteolytic systems hijacked by SARS-CoV for S protein activation, we will discuss their contribution to viral spread in the host and we will outline antiviral strategies targeting these enzymes. This paper forms part of a series of invited articles in Antiviral Research on "From SARS to MERS: 10years of research on highly pathogenic human coronaviruses.'' Copyright © 2013 Elsevier B.V. All rights reserved.
Directory of Open Access Journals (Sweden)
Michael Melesse
Full Text Available Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF complex or the anaphase-promoting complex (APC. Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20 in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell
Melesse, Michael; Choi, Eunyoung; Hall, Hana; Walsh, Michael J.; Geer, M. Ariel; Hall, Mark C.
2014-01-01
Regulated proteolysis mediated by the ubiquitin proteasome system is a fundamental and essential feature of the eukaryotic cell division cycle. Most proteins with cell cycle-regulated stability are targeted for degradation by one of two related ubiquitin ligases, the Skp1-cullin-F box protein (SCF) complex or the anaphase-promoting complex (APC). Here we describe an unconventional cell cycle-regulated proteolytic mechanism that acts on the Acm1 protein, an inhibitor of the APC activator Cdh1 in budding yeast. Although Acm1 can be recognized as a substrate by the Cdc20-activated APC (APCCdc20) in anaphase, APCCdc20 is neither necessary nor sufficient for complete Acm1 degradation at the end of mitosis. An APC-independent, but 26S proteasome-dependent, mechanism is sufficient for complete Acm1 clearance from late mitotic and G1 cells. Surprisingly, this mechanism appears distinct from the canonical ubiquitin targeting pathway, exhibiting several features of ubiquitin-independent proteasomal degradation. For example, Acm1 degradation in G1 requires neither lysine residues in Acm1 nor assembly of polyubiquitin chains. Acm1 was stabilized though by conditional inactivation of the ubiquitin activating enzyme Uba1, implying some requirement for the ubiquitin pathway, either direct or indirect. We identified an amino terminal predicted disordered region in Acm1 that contributes to its proteolysis in G1. Although ubiquitin-independent proteasome substrates have been described, Acm1 appears unique in that its sensitivity to this mechanism is strictly cell cycle-regulated via cyclin-dependent kinase (Cdk) phosphorylation. As a result, Acm1 expression is limited to the cell cycle window in which Cdk is active. We provide evidence that failure to eliminate Acm1 impairs activation of APCCdh1 at mitotic exit, justifying its strict regulation by cell cycle-dependent transcription and proteolytic mechanisms. Importantly, our results reveal that strict cell-cycle expression profiles
Shakerian, Mansour; Razavi, Seyed Hadi; Ziai, Seyed Ali; Khodaiyan, Faramarz; Yarmand, Mohammad Saeid; Moayedi, Ali
2013-01-01
In this study, the effects of fat (0.5 %, 3.2 % and 5.0 %), inulin (0.0 and 1.0 %) and starter culture (0.0 %, 0.5 %, 1.0 % and 1.5 %) on the angiotensin converting enzyme (ACE)-inhibitory activity of probiotic yogurt containing non-viable bacteria were assessed. Proteolytic activities of bacteria were also investigated. Yogurts were prepared either using a sole yogurt commercial culture including Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus or bifidobacterium ani...
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Farah El Najjar
Full Text Available Bats of different species play a major role in the emergence and transmission of highly pathogenic viruses including Ebola virus, SARS-like coronavirus and the henipaviruses. These viruses require proteolytic activation of surface envelope glycoproteins needed for entry, and cellular cathepsins have been shown to be involved in proteolysis of glycoproteins from these distinct virus families. Very little is currently known about the available proteases in bats. To determine whether the utilization of cathepsins by bat-borne viruses is related to the nature of proteases in their natural hosts, we examined proteolytic processing of several viral fusion proteins in cells derived from two fruit bat species, Pteropus alecto and Rousettus aegyptiacus. Our work shows that fruit bat cells have homologs of cathepsin and furin proteases capable of cleaving and activating both the cathepsin-dependent Hendra virus F and the furin-dependent parainfluenza virus 5 F proteins. Sequence analysis comparing Pteropus alecto furin and cathepsin L to proteases from other mammalian species showed a high degree of conservation; however significant amino acid variation occurs at the C-terminus of Pteropus alecto furin. Further analysis of furin-like proteases from fruit bats revealed that these proteases are catalytically active and resemble other mammalian furins in their response to a potent furin inhibitor. However, kinetic analysis suggests that differences may exist in the cellular localization of furin between different species. Collectively, these results indicate that the unusual role of cathepsin proteases in the life cycle of bat-borne viruses is not due to the lack of active furin-like proteases in these natural reservoir species; however, differences may exist between furin proteases present in fruit bats compared to furins in other mammalian species, and these differences may impact protease usage for viral glycoprotein processing.
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SARNER, NITZA Z; BISSELL, MINA J; GIROLAMO, MARIO Di; GORINI, LUIGI
1970-06-29
A Sarcina strain (Coccus P) produces two proteolytic enzymes. One is found only extracellularly, is far more prevalent, and is actively excreted during exponential growth. It is the enzyme responsible for the known strong proteolytic activity of the cultures of this strain. A second protease is, however, produced which remains associated with the intact cells but is released by the protoplasts. The two enzymes appear unrelated in their derivation. Calcium ions play an essential role in preventing autodigestion of the excreted enzyme. Bacterial proteins are found outside the cell boundary as a consequence either of passive processes such as leakage or lysis or of active excretion. Under conditions in which leakage and lysis do not occur, as during exponential growth, the cell boundary is a barrier causing a complete separation of the bulk of the intracellular proteins from the one or very few extracellular proteins, with no trace of either type being detectable on the wrong side of the boundary. Since in bacteria there is no evidence of protein being produced other than internally, the separation into intraand extracellular proteins should occur after peptide chain formation. The question arises as to whether the structure of the cell boundary or that of the excreted proteins themselves determines this separation. Coccus P, a Sarcina closely related to Micrococcus lysodeikticus (3), produces an extracellular proteinase during the exponential phase of growth so that the process appears to be active excretion. The organism grows exponentially in a defined synthetic medium (12) to relatively high cell density (10{sup 9} cells/ml); therefore the mechanism of excretion can be studied over an extended period of time without the difficulties of changing growth rates. Coagulation of reconstituted skim milk provides a simple and sensitive assay for enzyme activity (I 1). The extracellular proteinase has also been purified and partially characterized (6-8). It has been shown
International Nuclear Information System (INIS)
Pager, Cara Theresia; Craft, Willie Warren; Patch, Jared; Dutch, Rebecca Ellis
2006-01-01
The Nipah virus fusion (F) protein is proteolytically processed to F 1 + F 2 subunits. We demonstrate here that cathepsin L is involved in this important maturation event. Cathepsin inhibitors ablated cleavage of Nipah F. Proteolytic processing of Nipah F and fusion activity was dramatically reduced in cathepsin L shRNA-expressing Vero cells. Additionally, Nipah virus F-mediated fusion was inhibited in cathepsin L-deficient cells, but coexpression of cathepsin L restored fusion activity. Both purified cathepsin L and B could cleave immunopurified Nipah F protein, but only cathepsin L produced products of the correct size. Our results suggest that endosomal cathepsins can cleave Nipah F, but that cathepsin L specifically converts Nipah F to a mature and fusogenic form
Use of Proteolytic Enzymes in the Treatment of Proteinaceous Esophageal Food Impaction.
Morse, Christopher R; Wang, Howard; Donahue, Dean M; Garrity, Julie M; Allan, James S
2016-01-01
Proteinaceous esophageal food impaction typically requires endoscopic intervention. An alternative approach is the use of proteolytic enzymes. Concerns regarding the use of proteolytic enzymes include the risk of perforation and aspiration pneumonitis. We retrospectively reviewed our series of 69 patients treated with papain to determine the safety and efficacy of proteolytic enzymes. Patients were retrospectively reviewed if treated for an esophageal food impaction from 1999 through 2008. Median age was 56 years (range 19-91 years), with 46 male and 23 female patients. In 27 patients (39%) this was their first presentation, in 14 (20%) it was the second, and 28 (41%) had multiple previous episodes. Meat was the cause in 49 (71%), chicken in 6 (9%), fish in 3 (4%), and unspecified in 11 (16%). All patients presented with dysphagia for solids, 56 (81%) could not tolerate liquids. Papain solution, 1 tsp in 8 oz of water, was given to patients in an unlimited quantity. Papain was successful in relieving the obstruction in 60 patients (87%). The remaining 9 patients (13%) underwent endoscopy with successful retrieval. No patient suffered a perforation, either with papain ingestion or endoscopy. There were no episodes of pneumonitis or pneumonia. We have used proteolytic enzymes with a high success rate and with minimal complication. Further, if proteolytic enzymes fail, endoscopy can be performed safely and effectively. We recommend the use of proteolytic enzymes as the initial management in all patients with proteinaceous food impaction of the esophagus. Published by Elsevier Inc.
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Mauro Manuel Martinez-Pacheco
2011-10-01
Full Text Available Hybrids of Zea mays L. (Buffalo, Falcon, H360 y HV313 were treated with citric acid (2000 ppm. Grain yield, soluble protein and proteolytic activity were monitored when the crop reached physiological maturity. Citric acid was applied before the appearance of the flag leaf, and induced an increase in the grain yield from 4222 to 5780 kg/ha, in the soluble protein from 6.34 to 7.91 mg/mg and into the proteolytic activity from 14.3 to 65.7 µU mg prot-1. This is an increase of 2 to 12 times in the Falcon, H360 and HV313 hybrids, while the Buffalo hybrid responded with less intensity to the treatment with citric acid. In the H360 hybrid treated with citric acid, an increase in the proteolytic activity of aspartyl serine proteases was observed. The results indicate that citric acid differentially induces proteolytic activity and vigor in the corn hybrids analyzed.Zea mays L. híbridos (Buffalo, Falcão, H360 e HV313 foram tratados com ácido cítrico (2000 ppm. O rendimento de grãos, proteína solúvel e atividade proteolítica foram monitorados na fase de maturação fisiológica do cultivo. O ácido cítrico aplicado antes do aparecimento da folha bandeira induziu um aumento na produção de grãos 4222 a 5780 kg/ha, na proteína solúvel de 6.34 7.91 mg/mg de peso seco e atividade proteolítica de 14.3 to 65.7 µU mg prot-1, esto é, um incremento de 2-12 vezes nos híbridos Falcao, H360 e HV313, enquanto o híbrido Buffalo responderam com menor intensidade ao tratamento com ácido cítrico. No híbrido H360, tratadas com ácido cítrico, apresentou-se um aumento na atividade proteolítica de aspartil proteases e serin protease. Os resultados indicam que o ácido cítrico diferencialmente induz a atividade proteolítica e o vigor de híbridos de milho testados.
[Treatment of surface burns with proteolytic enzymes: mathematic description of lysis kinetics].
Domogatskaia, A S; Domogatskiĭ, S P; Ruuge, E K
2003-01-01
The lysis of necrotic tissue by a proteolytic enzyme applied to the surface of a burn wound was studied. A mathematical model was proposed, which describes changes in the thickness of necrotic tissue as a function of the proteolytic activity of the enzyme. The model takes into account the inward-directed diffusion of the enzyme, the counterflow of interstitial fluid (exudates) containing specific inhibitors, and the extracellular matrix proteolysis. It was shown in terms of the quasi-stationary approach that the thickness of the necrotic tissue layer decreases exponentially with time; i.e., the lysis slows down as the thickness of the necrotic tissue layer decreases. The dependence of the characteristic time of this decrease on enzyme concentration was obtained. It was shown that, at high enzyme concentrations (more than 5 mg/ml), the entire time of lysis (after the establishment of quasi-stationary equilibrium) is inversely proportional to the concentration of the enzyme.
Detection of proteolytic signatures for Parkinson's disease
DEFF Research Database (Denmark)
Jordal, Peter Lüttge; Dyrlund, Thomas F.; Winge, Kristian
2016-01-01
Aim: To investigate if idiopathic Parkinson's disease (IPD) is associated with distinct proteolytic signatures relative to non-neurodegenerative controls (NND) and patients with multiple system atrophy (MSA). Materials & methods: A subtiligase-based N-terminomics screening method was exploited...
The proteolytic systems of lactic acid bacteria
Kunji, Edmund R.S.; Mierau, Igor; Hagting, Anja; Poolman, Bert; Konings, Wil N.
1996-01-01
Proteolysis in dairy lactic acid bacteria has been studied in great detail by genetic, biochemical and ultrastructural methods. From these studies the picture emerges that the proteolytic systems of lactococci and lactobacilli are remarkably similar in their components and mode of action. The
Bicarbonate-dependent secretion and proteolytic processing of recombinant myocilin.
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José-Daniel Aroca-Aguilar
Full Text Available Myocilin is an extracellular glycoprotein of poorly understood function. Mutations of this protein are involved in glaucoma, an optic neuropathy characterized by a progressive and irreversible visual loss and frequently associated with elevated intraocular pressure. We previously showed that recombinant myocilin undergoes an intracellular proteolytic processing by calpain II which cleaves the central region of the protein, releasing one N- and one C-terminal fragment. Myocilin cleavage is reduced by glaucoma mutations and it has been proposed to participate in intraocular pressure modulation. To identify possible factors regulating the proteolytic processing of recombinant myocilin, we used a cellular model in which we analyzed how different culture medium parameters (i.e., culture time, cell density, pH, bicarbonate concentration, etc. affect the presence of the extracellular C-terminal fragment. Extracellular bicarbonate depletion associated with culture medium acidification produced a reversible intracellular accumulation of full-length recombinant myocilin and incremented its intracellular proteolytic processing, raising the extracellular C-terminal fragment percentage. It was also determined that myocilin intracellular accumulation depends on its N-terminal region. These data suggest that aqueous humor bicarbonate variations could also modulate the secretion and cleavage of myocilin present in ocular tissues.
Comparing Proteolytic Fingerprints of Antigen-Presenting Cells during Allergen Processing.
Hofer, Heidi; Weidinger, Tamara; Briza, Peter; Asam, Claudia; Wolf, Martin; Twaroch, Teresa E; Stolz, Frank; Neubauer, Angela; Dall, Elfriede; Hammerl, Peter; Jacquet, Alain; Wallner, Michael
2017-06-08
Endolysosomal processing has a critical influence on immunogenicity as well as immune polarization of protein antigens. In industrialized countries, allergies affect around 25% of the population. For the rational design of protein-based allergy therapeutics for immunotherapy, a good knowledge of T cell-reactive regions on allergens is required. Thus, we sought to analyze endolysosomal degradation patterns of inhalant allergens. Four major allergens from ragweed, birch, as well as house dust mites were produced as recombinant proteins. Endolysosomal proteases were purified by differential centrifugation from dendritic cells, macrophages, and B cells, and combined with allergens for proteolytic processing. Thereafter, endolysosomal proteolysis was monitored by protein gel electrophoresis and mass spectrometry. We found that the overall proteolytic activity of specific endolysosomal fractions differed substantially, whereas the degradation patterns of the four model allergens obtained with the different proteases were extremely similar. Moreover, previously identified T cell epitopes were assigned to endolysosomal peptides and indeed showed a good overlap with known T cell epitopes for all four candidate allergens. Thus, we propose that the degradome assay can be used as a predictor to determine antigenic peptides as potential T cell epitopes, which will help in the rational design of protein-based allergy vaccine candidates.
DEFF Research Database (Denmark)
Søe, Rikke; Overgaard, Michael Toft; Thomsen, Anni R
2002-01-01
Murine pregnancy-associated plasma protein-A (PAPP-A) cDNA encoding a 1545 amino-acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP-A, PAPP-Ai, carrying a 29-residue highly basic insert. The point of insertion corresponds to a junction between...... with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP-A-proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP-Ai and the linkage to proMBP is not known. Importantly, these data...
Wang, Liwei; Alzayady, Kamil J; Yule, David I
2016-06-01
Inositol 1,4,5-trisphosphate receptors (IP3 Rs) are a family of ubiquitously expressed intracellular Ca(2+) release channels. Regulation of channel activity by Ca(2+) , nucleotides, phosphorylation, protein binding partners and other cellular factors is thought to play a major role in defining the specific spatiotemporal characteristics of intracellular Ca(2+) signals. These properties are, in turn, believed pivotal for the selective and specific physiological activation of Ca(2+) -dependent effectors. IP3 Rs are also substrates for the intracellular cysteine proteases, calpain and caspase. Cleavage of the IP3 R has been proposed to play a role in apoptotic cell death by uncoupling regions important for IP3 binding from the channel domain, leaving an unregulated leaky Ca(2+) pore. Contrary to this hypothesis, we demonstrate following proteolysis that N- and C-termini of IP3 R1 remain associated, presumably through non-covalent interactions. Further, we show that complementary fragments of IP3 R1 assemble into tetrameric structures and retain their ability to be regulated robustly by IP3 . While peptide continuity is clearly not necessary for IP3 -gating of the channel, we propose that cleavage of the IP3 R peptide chain may alter other important regulatory events to modulate channel activity. In this scenario, stimulation of the cleaved IP3 R may support distinct spatiotemporal Ca(2+) signals and activation of specific effectors. Notably, in many adaptive physiological events, the non-apoptotic activities of caspase and calpain are demonstrated to be important, but the substrates of the proteases are poorly defined. We speculate that proteolytic fragmentation may represent a novel form of IP3 R regulation, which plays a role in varied adaptive physiological processes. © 2015 The Authors. The Journal of Physiology © 2015 The Physiological Society.
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Latchoumycandane, Calivarathan; Anantharam, Vellareddy; Jin, Huajun; Kanthasamy, Anumantha; Kanthasamy, Arthi, E-mail: arthik@iastate.edu
2011-11-15
The neurotoxicant 6-hydroxydopamine (6-OHDA) is used to investigate the cellular and molecular mechanisms underlying selective degeneration of dopaminergic neurons in Parkinson's disease (PD). Oxidative stress and caspase activation contribute to the 6-OHDA-induced apoptotic cell death of dopaminergic neurons. In the present study, we sought to systematically characterize the key downstream signaling molecule involved in 6-OHDA-induced dopaminergic degeneration in cell culture and animal models of PD. Treatment of mesencephalic dopaminergic neuronal N27 cells with 6-OHDA (100 {mu}M) for 24 h significantly reduced mitochondrial activity and increased cytosolic cytochrome c, followed by sequential activation of caspase-9 and caspase-3. Co-treatment with the free radical scavenger MnTBAP (10 {mu}M) significantly attenuated 6-OHDA-induced caspase activities. Interestingly, 6-OHDA induced proteolytic cleavage and activation of protein kinase C delta (PKC{delta}) was completely suppressed by treatment with a caspase-3-specific inhibitor, Z-DEVD-FMK (50 {mu}M). Furthermore, expression of caspase-3 cleavage site-resistant mutant PKC{delta}{sup D327A} and kinase dead PKC{delta}{sup K376R} or siRNA-mediated knockdown of PKC{delta} protected against 6-OHDA-induced neuronal cell death, suggesting that caspase-3-dependent PKC{delta} promotes oxidative stress-induced dopaminergic degeneration. Suppression of PKC{delta} expression by siRNA also effectively protected N27 cells from 6-OHDA-induced apoptotic cell death. PKC{delta} cleavage was also observed in the substantia nigra of 6-OHDA-injected C57 black mice but not in control animals. Viral-mediated delivery of PKC{delta}{sup D327A} protein protected against 6-OHDA-induced PKC{delta} activation in mouse substantia nigra. Collectively, these results strongly suggest that proteolytic activation of PKC{delta} is a key downstream event in dopaminergic degeneration, and these results may have important translational value for
The role of lysosomal proteolytic enzymes in invasion and dissemination of malignant melanoma
International Nuclear Information System (INIS)
Bassalyk, L.S.; Tsanev, P.E.; Parshikova, S.M.; Demidov, L.V.
1992-01-01
Preoperative chemo- and radiation therapy was followed by a decrease in lysosomal cathepsins activity in metastatic lymph nodes which, however, did not reach the level established for intact lymph nodes. The pathogenetic role of proteolytic endopeptidases in invasion and sissemination of malignant melanoma is discussed as well as the value of their level measurement for assessing metastatic potential of tumor and prognosis of disease of disease on the basis of tumor site, degree of invasion regional lymph node status
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Yu Wei-Hsuan
2012-05-01
Full Text Available Abstract Background The Aim of this study is to study the minimum zinc dependent metalloprotease catalytic folding motif, helix B Met loop-helix C, with proteolytic catalytic activities in metzincin super family. The metzincin super family share a catalytic domain consisting of a twisted five-stranded β sheet and three long α helices (A, B and C. The catalytic zinc is at the bottom of the cleft and is ligated by three His residues in the consensus sequence motif, HEXXHXXGXXH, which is located in helix B and part of the adjacent Met turn region. An interesting question is - what is the minimum portion of the enzyme that still possesses catalytic and inhibitor recognition?” Methods We have expressed a 60-residue truncated form of matrilysin which retains only the helix B-Met turn-helix C region and deletes helix A and the five-stranded β sheet which form the upper portion of the active cleft. This is only 1/4 of the full catalytic domain. The E. coli derived 6 kDa MMP-7 ZBD fragments were purified and refolded. The proteolytic activities were analyzed by Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay and CM-transferrin zymography analysis. SC44463, BB94 and Phosphoramidon were computationally docked into the 3day structure of the human MMP7 ZBD and TAD and thermolysin using the docking program GOLD. Results This minimal 6 kDa matrilysin has been refolded and shown to have proteolytic activity in the Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 peptide assay. Triton X-100 and heparin are important factors in the refolding environment for this mini-enzyme matrilysin. This minienzyme has the proteolytic activity towards peptide substrate, but the hexamer and octamer of the mini MMP-7 complex demonstrates the CM-transferrin proteolytic activities in zymographic analysis. Peptide digestion is inhibited by SC44463, specific MMP7 inhibitors, but not phosphorimadon. Interestingly, the mini MMP-7 can be processed by autolysis and producing ~ 6
An, Young Jun; Na, Jung-Hyun; Kim, Myung-Il; Cha, Sun-Shin
2015-10-01
Lon proteases degrade defective or denature proteins as well as some folded proteins for the control of cellular protein quality. There are two types of Lon proteases, LonA and LonB. Each consists of two functional components: a protease component and an ATPase associated with various cellular activities (AAA+ module). Here, we report the 2.03 -resolution crystal structure of the isolated AAA+ module (iAAA+ module) of LonB from Thermococcus onnurineus NA1 (TonLonB). The iAAA+ module, having no bound nucleotide, adopts a conformation virtually identical to the ADP-bound conformation of AAA+ modules in the hexameric structure of TonLonB; this provides insights into the ATP-independent proteolytic activity observed in a LonB protease. Structural comparison of AAA+ modules between LonA and LonB revealed that the AAA+ modules of Lon proteases are separated into two distinct clades depending on their structural features. The AAA+ module of LonB belongs to the -H2 & Ins1 insert clade (HINS clade)- defined for the first time in this study, while the AAA+ module of LonA is a member of the HCLR clade.
Biziulevicius, Gediminas A
2006-01-01
Enteric-coated proteolytic enzyme preparations like Wobenzym and Phlogenzym are widely used for the so-called 'systemic enzyme therapy' both in humans and animals. Numerous publications reveal that oral proteolytic enzymes are able to stimulate directly the activity of immune competent cells as well as to increase efficiency of some of their products. But origins of the immunostimulatory effects of oral proteolytic enzymes are still unclear. The hypothesis described here suggests that it may be proteolysis of intestinal microorganisms that makes the immune competent cells to work in the immunostimulatory manner. The hypothesis was largely formed by several scientific observations: First, microbial lysis products (lipopolysaccharides, muropeptides and other peptidoglycan fragments, beta-glucans, etc.) are well known for their immunostimulatory action. Second, a normal human being hosts a mass of intestinal microorganisms equivalent to about 1 kg. The biomass (mainly due to naturally occurring autolysis) continuously supplies the host's organism with immunostimulatory microbial cell components. Third, the immunostimulatory effects resulting from the oral application of exogenously acting antimicrobial (lytic) enzyme preparations, such as lysozyme and lysosubtilin, are likely to be a result of the action of microbial lysis products. Fourth, cell walls of most microorganisms contain a considerable amount of proteins/peptides, a possible target for exogenous proteolytic enzymes. In fact, several authors have already shown that a number of proteases possess an ability to lyse the microbial cells in vitro. Fifth, the pretreatment of microbial cells (at least of some species) in vitro with proteolytic enzymes makes them more sensitive to the lytic action of lysozyme and, otherwise, pretreatment with lysozyme makes them more susceptible to proteolytic degradation. Sixth, exogenous proteases, when in the intestines, may participate in final steps of food-protein digestion
A radiolabel-release microwell assay for proteolytic enzymes present in cell culture media
International Nuclear Information System (INIS)
Rucklidge, G.J.; Milne, G.
1990-01-01
A modified method for the measurement of proteolytic enzyme activity in cell culture-conditioned media has been developed. Using the release of 3H-labeled peptides from 3H-labeled gelatin the method is performed in microwell plates. The substrate is insolubilized and attached to the wells by glutaraldehyde treatment, thus eliminating the need for a precipitation step at the end of the assay. The assay is sensitive, reproducible, and convenient for small sample volumes. The effect of different protease inhibitors on activity can be assessed rapidly allowing an early characterization of the enzyme. It can also be adapted to microplate spectrophotometric analysis by staining residual substrate with Coomassie blue
Factors affecting proteolytic action of Lactococcus lactis in cheese
Youssef, Y.B.
1992-01-01
Model cheeses were developed to study the behaviour of proteolytic agents involved in cheese maturation under conditions that closely resemble those in normal cheese. The models were applied to study protein breakdown by Lactococcus lactis ssp. cremoris HP , as a
Site-specific proteolytic degradation of IgG monoclonal antibodies expressed in tobacco plants.
Hehle, Verena K; Lombardi, Raffaele; van Dolleweerd, Craig J; Paul, Mathew J; Di Micco, Patrizio; Morea, Veronica; Benvenuto, Eugenio; Donini, Marcello; Ma, Julian K-C
2015-02-01
Plants are promising hosts for the production of monoclonal antibodies (mAbs). However, proteolytic degradation of antibodies produced both in stable transgenic plants and using transient expression systems is still a major issue for efficient high-yield recombinant protein accumulation. In this work, we have performed a detailed study of the degradation profiles of two human IgG1 mAbs produced in plants: an anti-HIV mAb 2G12 and a tumour-targeting mAb H10. Even though they use different light chains (κ and λ, respectively), the fragmentation pattern of both antibodies was similar. The majority of Ig fragments result from proteolytic degradation, but there are only a limited number of plant proteolytic cleavage events in the immunoglobulin light and heavy chains. All of the cleavage sites identified were in the proximity of interdomain regions and occurred at each interdomain site, with the exception of the VL /CL interface in mAb H10 λ light chain. Cleavage site sequences were analysed, and residue patterns characteristic of proteolytic enzymes substrates were identified. The results of this work help to define common degradation events in plant-produced mAbs and raise the possibility of predicting antibody degradation patterns 'a priori' and designing novel stabilization strategies by site-specific mutagenesis. © 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.
Lü, Fan; Bize, Ariane; Guillot, Alain; Monnet, Véronique; Madigou, Céline; Chapleur, Olivier; Mazéas, Laurent; He, Pinjing; Bouchez, Théodore
2014-01-01
Cellulose is the most abundant biopolymer on Earth. Optimising energy recovery from this renewable but recalcitrant material is a key issue. The metaproteome expressed by thermophilic communities during cellulose anaerobic digestion was investigated in microcosms. By multiplying the analytical replicates (65 protein fractions analysed by MS/MS) and relying solely on public protein databases, more than 500 non-redundant protein functions were identified. The taxonomic community structure as inferred from the metaproteomic data set was in good overall agreement with 16S rRNA gene tag pyrosequencing and fluorescent in situ hybridisation analyses. Numerous functions related to cellulose and hemicellulose hydrolysis and fermentation catalysed by bacteria related to Caldicellulosiruptor spp. and Clostridium thermocellum were retrieved, indicating their key role in the cellulose-degradation process and also suggesting their complementary action. Despite the abundance of acetate as a major fermentation product, key methanogenesis enzymes from the acetoclastic pathway were not detected. In contrast, enzymes from the hydrogenotrophic pathway affiliated to Methanothermobacter were almost exclusively identified for methanogenesis, suggesting a syntrophic acetate oxidation process coupled to hydrogenotrophic methanogenesis. Isotopic analyses confirmed the high dominance of the hydrogenotrophic methanogenesis. Very surprising was the identification of an abundant proteolytic activity from Coprothermobacter proteolyticus strains, probably acting as scavenger and/or predator performing proteolysis and fermentation. Metaproteomics thus appeared as an efficient tool to unravel and characterise metabolic networks as well as ecological interactions during methanisation bioprocesses. More generally, metaproteomics provides direct functional insights at a limited cost, and its attractiveness should increase in the future as sequence databases are growing exponentially.
A plant Bcl-2-associated athanogene is proteolytically activated to confer fungal resistance
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Mehdi Kabbage
2016-04-01
Full Text Available The Bcl-2-associated athanogene (BAG family is a multifunctional group of proteins involved in numerous cellular functions ranging from apoptosis to tumorigenesis. These proteins are evolutionarily conserved and encode a characteristic region known as the BAG domain. BAGs function as adapter proteins forming complexes with signaling molecules and molecular chaperones. In humans, a role for BAG proteins has been suggested in tumor growth, HIV infection, and neurodegenerative diseases; as a result, the BAGs are attractive targets for therapeutic interventions, and their expression in cells may serve as a predictive tool for disease development. The Arabidopsis genome contains seven homologs of BAG family proteins (Figure 1, including four with a domain organization similar to animal BAGs (BAG1-4. The remaining three members (BAG5-7 contain a predicted calmodulin-binding motif near the BAG domain, a feature unique to plant BAG proteins that possibly reflects divergent mechanisms associated with plant-specific functions. As reported for animal BAGs, plant BAGs also regulate several stress and developmental processes (Figure 2. The recent article by Li et al. focuses on the role of BAG6 in plant innate immunity. This study shows that BAG6 plays a key role in basal plant defense against fungal pathogens. Importantly, this work further shows that BAG6 is proteolytically activated to induce autophagic cell death and resistance in plants. This finding underscores the importance of proteases in the execution of plant cell death, yet little is known about proteases and their substrates in plants.
Yang, Fang; Rustad, Turid; Xu, Yanshun; Jiang, Qixing; Xia, Wenshui
2015-04-01
The aim of this study was to investigate endogenous proteolytic activities in a cod product and their impact on muscle proteins and textural properties during fermentation and storage. The result of specific proteolytic activities showed that cathepsins, especially cathepsin B, had the highest activities during fermentation and storage. SDS-PAGE indicated more degradation of myofibrillar proteins by cathepsin L than other proteases and that the hydrolysis by cathepsins was pronounced in the last stage of fermentation. Texture analysis showed that cathepsins had a negative impact on gel strength and this impact increased in the last stage of fermentation. However the product still had a firm texture. During storage (4 °C) for one week, no significant changes were seen in the gel strength. In conclusion, cathepsins had more impact on muscle proteins and textural properties than other proteases during fermentation but had little impact on gel strength during storage at 4 °C. Copyright © 2014 Elsevier Ltd. All rights reserved.
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Joseph Chavarría-Smith
Full Text Available Inflammasomes are multimeric protein complexes that respond to infection by recruitment and activation of the Caspase-1 (CASP1 protease. Activated CASP1 initiates immune defense by processing inflammatory cytokines and by causing a rapid and lytic cell death called pyroptosis. Inflammasome formation is orchestrated by members of the nucleotide-binding domain and leucine-rich repeat (NLR or AIM2-like receptor (ALR protein families. Certain NLRs and ALRs have been shown to function as direct receptors for specific microbial ligands, such as flagellin or DNA, but the molecular mechanism responsible for activation of most NLRs is still poorly understood. Here we determine the mechanism of activation of the NLRP1B inflammasome in mice. NLRP1B, and its ortholog in rats, is activated by the lethal factor (LF protease that is a key virulence factor secreted by Bacillus anthracis, the causative agent of anthrax. LF was recently shown to cleave mouse and rat NLRP1 directly. However, it is unclear if cleavage is sufficient for NLRP1 activation. Indeed, other LF-induced cellular events have been suggested to play a role in NLRP1B activation. Surprisingly, we show that direct cleavage of NLRP1B is sufficient to induce inflammasome activation in the absence of LF. Our results therefore rule out the need for other LF-dependent cellular effects in activation of NLRP1B. We therefore propose that NLRP1 functions primarily as a sensor of protease activity and thus could conceivably detect a broader spectrum of pathogens than just B. anthracis. By adding proteolytic cleavage to the previously established ligand-receptor mechanism of NLR activation, our results illustrate the remarkable flexibility with which the NLR architecture can be deployed for the purpose of pathogen-detection and host defense.
Genetics of the proteolytic system of lactic acid bacteria
Kok, Jan
1990-01-01
The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have
International Nuclear Information System (INIS)
Ramanathan, Mathura P.; Chambers, Jerome A.; Pankhong, Panyupa; Chattergoon, Michael; Attatippaholkun, Watcharee; Dang, Kesen; Shah, Neelima; Weiner, David B.
2006-01-01
The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the apoptotic pathways involving caspases-8 and -3. Experimental results from the use of caspase-specific inhibitors and caspase-8 siRNA demonstrated that the activation of caspase-8 was essential to initiate apoptotic signaling in NS3-expressing cells. Downstream of caspase-3 activation, we observed nuclear membrane ruptures and cleavage of the DNA-repair enzyme, PARP in NS3-expressing cells. Nuclear herniations due to NS3 expression were absent in the cells treated with a caspase-3 inhibitor. Expression of protease and helicase domains themselves was sufficient to trigger apoptosis generating insight into the apoptotic pathways triggered by NS3 from WNV
[Inhibitors of proteolytic enzymes under abiotic stresses in plants (review)].
Mosolov, V V; Valueva, T A
2011-01-01
Data on the role of proteolytic enzyme inhibitors in plant adaptation to various unfavorable environmental abiotic factors--water deficiency, salinization of soil, extreme temperatures, etc.--and also probable functions of proteinases inhibitors in natural plant senescense are considered.
Deng, Jingren; Lazar, Iulia M.
2016-04-01
The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples.
Winnick, Theodore; Davis, Alva R.; Greenberg, David M.
1940-01-01
1. A study has been made of the properties of a hitherto unreported proteolytic enzyme from the latex of the milkweed, Asclepias speciosa. The new protease has been named asclepain by the authors. 2. The results of chemical, diffusion, and denaturation tests indicate that asclepain is a protein. 3. Like papain, asclepain dots milk and digests most proteins, particularly if they are dissolved in concentrated urea solution. Unlike papain, asclepain did not clot blood. 4. The activation and inhibition phenomena of asclepain resemble those of papain, and seem best explained on the assumption that free sulfhydryl in the enzyme is necessary for proteolytic activity. The sulfhydryl of asclepain appears more labile than that of papain. 5. The measurement of pH-activity curves of asclepain on casein, ovalbumin, hemoglobin, edestin, and ovovitellin showed no definite digestion maxima for most of the undenatured proteins, while in urea solution there were well defined maxima near pH 7.0. Native hemoglobin and ovovitellin were especially undigestible, while native casein was rapidly attacked. 6. Temperature-activity curves were determined for asclepain on hemoglobin, casein, and milk solutions. The optimum temperature was shown to increase with decreasing time of digestion. PMID:19873154
Effects of Sunphenon and Polyphenon 60 on proteolytic pathways ...
Indian Academy of Sciences (India)
2016-08-26
Aug 26, 2016 ... The effect of Sunphenon and Polyphenon 60 in oxidative stress response, myogenic regulatory factors, inflammatory cytokines, apoptotic and proteolytic pathways on H2O2-induced myotube atrophy was addressed. Cellular responses of H2O2-induced C2C12cells were examined, including mRNA ...
Production of Proteolytic Enzymes by a Keratin-Degrading Aspergillus niger
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Fernanda Cortez Lopes
2011-01-01
Full Text Available A fungal isolate with capability to grow in keratinous substrate as only source of carbon and nitrogen was identified as Aspergillus niger using the sequencing of the ITS region of the rDNA. This strain produced a slightly acid keratinase and an acid protease during cultivation in feather meal. The peak of keratinolytic activity occurred in 48 h and the maximum proteolytic activity in 96 h. These enzymes were partly characterized as serine protease and aspartic protease, respectively. The effects of feather meal concentration and initial pH on enzyme production were evaluated using a central composite design combined with response surface methodology. The optimal conditions were determined as pH 5.0 for protease and 7.8 for keratinase and 20 g/L of feather meal, showing that both models were predictive. Production of keratinases by A. niger is a less-exploited field that might represent a novel and promising biotechnological application for this microorganism.
Hemmer, O; Greif, C; Dufourcq, P; Reinbolt, J; Fritsch, C
1995-01-10
Translation of tomato black ring virus (TBRV) RNA-1 in a rabbit reticulocyte lysate leads to the synthesis of a 250K polyprotein which cleaves itself into smaller proteins of 50, 60, 120, and 190K. Polypeptides synthesized from synthetic transcripts corresponding to different regions of TBRV RNA-1 are processed only when they encode the 23K protein delimited earlier by sequence homology with the cowpea mosaic virus 24K protease. The proteolytic activity of this protein is completely lost by mutating residues C170 (to I) or L188 (to H), residues which align with conserved residues of the viral serine-like proteases. The 120K protein is generated by cleavage of the dipeptide K/A localized in front of the VPg but is not further cleaved in vitro at the K/S site (at the C terminus of the VPg) or between the protease and polymerase domains. However, both the protein VPgProPol (120K) and the protein ProPol (117K) produced in vitro from synthetic transcripts can cleave in trans the RNA-2-encoded 150K polyprotein, but they cannot cleave in trans polypeptides containing a cleavage site expressed from RNA-1 transcripts in which the protease cistron is absent or modified.
Institute of Scientific and Technical Information of China (English)
XIE Xiao-mei; LIAO Min; LIU Wei-ping; Susanne KLOSE
2004-01-01
Combined effects on soil microbial activity of nutrient and pesticide management in hybrid rice double annual cropping system were studied. Results of field experiment demonstrated significant changes in soil microbial biomass phospholipid contents,abundance of heterotrophic bacteria and proteolytic bacteria, electron transport system (ETS)/dehydrogenase activity, soil protein contents under different management practices and at various growth stages. Marked depletions in the soil microbial biomass phospholipid contents were found with the advancement of crop growth stages, while the incorporation of fertilizers and/or pesticides also induced slight changes, and the lowest microbial biomass phospholipid content was found with pesticides application alone. A decline in the bacterial abundance of heterotrophic bacteria and proteolytic bacteria was observed during the continuance of crop growth, while the lowest abundance of heterotrophic bacteria and proteolytic bacteria was found with pesticides application alone, which coincided with the decline of soil microbial biomass. A consistent increase in the electron transport system activity was measured during the different crop growth stages of rice. The use of fertilizers (NPK) alone or combined with pesticides increased it, while a decline was noticed with pesticides application alone as compared with the control.The soil protein content was found to be relatively stable with fertilizers and/or pesticides application at various growth stages in both crops undertaken, but notable changes were detected at different growth stages.
Directory of Open Access Journals (Sweden)
Fabio Ribeiro Braga
Full Text Available The objective of this study was to examine the action of the crude extract of Duddingtonia flagrans (isolates AC001 and CG722 on infective larvae (L3 of cyathostomins in coprocultures and to confirm its proteolytic activity by means of a zymogram. The following groups were formed in coprocultures: Group 1: 10 mL of crude extract of D. flagrans (AC001; group 2: 10 mL of crude extract of AC001 with 10 mM of Ca2+; group 3: 10 mL of crude extract of D. flagrans (CG722; group 4: 10 mL of crude extract of CG722 with 10 mM of Ca2+; and group 5: control group (distilled water. The third-stage larvae (L3 were obtained after eight days. The crude extract of D. flagrans was effective in reducing the number of L3, with the following percentage reductions: group 1, 49.5%; group 2, 52.5%; group 3, 36.8%; and group 4, 57.7%; in relation to the control group (p > 0.05. The proteolytic activity of the crude extract was confirmed through the zymogram. The results from this study confirmed that the crude extract of the fungusD. flagrans could be used for controlling cyathostomin L3, and suggested that at least one protease of approximately 38 kDa was present.
Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum.
Carter, Andrew T; Paul, Catherine J; Mason, David R; Twine, Susan M; Alston, Mark J; Logan, Susan M; Austin, John W; Peck, Michael W
2009-03-19
Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs) present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes), and the flagellar glycosylation island (FGI). These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5) has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism outbreaks.
Independent evolution of neurotoxin and flagellar genetic loci in proteolytic Clostridium botulinum
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Twine Susan M
2009-03-01
Full Text Available Abstract Background Proteolytic Clostridium botulinum is the causative agent of botulism, a severe neuroparalytic illness. Given the severity of botulism, surprisingly little is known of the population structure, biology, phylogeny or evolution of C. botulinum. The recent determination of the genome sequence of C. botulinum has allowed comparative genomic indexing using a DNA microarray. Results Whole genome microarray analysis revealed that 63% of the coding sequences (CDSs present in reference strain ATCC 3502 were common to all 61 widely-representative strains of proteolytic C. botulinum and the closely related C. sporogenes tested. This indicates a relatively stable genome. There was, however, evidence for recombination and genetic exchange, in particular within the neurotoxin gene and cluster (including transfer of neurotoxin genes to C. sporogenes, and the flagellar glycosylation island (FGI. These two loci appear to have evolved independently from each other, and from the remainder of the genetic complement. A number of strains were atypical; for example, while 10 out of 14 strains that formed type A1 toxin gave almost identical profiles in whole genome, neurotoxin cluster and FGI analyses, the other four strains showed divergent properties. Furthermore, a new neurotoxin sub-type (A5 has been discovered in strains from heroin-associated wound botulism cases. For the first time, differences in glycosylation profiles of the flagella could be linked to differences in the gene content of the FGI. Conclusion Proteolytic C. botulinum has a stable genome backbone containing specific regions of genetic heterogeneity. These include the neurotoxin gene cluster and the FGI, each having evolved independently of each other and the remainder of the genetic complement. Analysis of these genetic components provides a high degree of discrimination of strains of proteolytic C. botulinum, and is suitable for clinical and forensic investigations of botulism
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Jose Carlos Ribeiro Junior
2015-12-01
Full Text Available The shelf life of milk and milk derivatives is directly related to the microbiological quality of refrigerated raw milk. Spoilage microorganisms with proteolytic and/or lipolytic properties are primarily responsible for the decrease in the quality of milk, which is reflected in the shelf life of pasteurized milk and all derivatives. The aim of this study was to determine the spoilage microbial load of refrigerated raw milk from the northeast and southern regions of Brazil, which have different climatic and technological conditions of production. We evaluated 46 samples of milk from the state of Paraná in the southern region, and 10 samples of milk from the state of Maranhão in the northeast region, totaling 56 samples collected from November 2013 to November 2014. The producers of Paraná were divided into large (20 or small (26 according to the average daily production. All producers of Maranhão were considered small (<500L/day. The proteolytic and lipolytic microorganism counts were conducted in milk agar and tributyrin agar, respectively. Milk from the large producers of Paraná had average counts of 1.4 × 104 CFU/mL for proteolytic microorganisms and 1.2 × 103 CFU/mL for lipolytics microorganisms, significantly (p <0.05 lower than the small producers in the same state, and the producers of Maranhão. Producers of Maranhao had counts of 1.1 × 105 CFU/mL for proteolytic microorganisms and 2 × 105 CFU/mL for lipolytic microorganisms, with the proteolytic count significantly lower than that of small Paraná producers. The amount of proteolytic and lipolytic spoilage microorganisms in milk is influenced by the adaptation of the microorganisms to cold, promoted by the cooling of milk, which is practiced less frequently in the country’s northeastern region. The amount of spoilage microorganisms is also affected by the implementation of milking hygiene practices, which reduce contamination. Such practices are more frequently and efficiently
Singh, Raja B; Hryshko, Larry; Freed, Darren; Dhalla, Naranjan S
2012-02-01
We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.
Zhylyak, Gleb; Ramoz-Perez, Victor; Linnhoff, Michael; Hug, Thomas; Citterio, Daniel; Spichiger-Keller, Ursula E.
2005-03-01
The paper shows the very first results of a feasibility study where the activity of proteolytic enzymes towards dye-labelled artificial substrates immobilized on the surface of planar optical Ta2O5 waveguide was investigated. Within this project, a chromophore label was developed, synthesized and attached to the carboxy-terminus of specific tripeptides. The goal was to develop a highly sensitive optical assay in order to monitor the activity of serine-proteases by cleavage of the amide bond between peptide and chromophore. On the one hand, a strategy was developed to immobilize the labeled tripeptide unto integrated planar waveguides. On the other hand, an instrument, the so-called "chip-reader" was developed to detect the biological process on the surface of the integrated planar optical waveguide. Surface characteristics were analyzed by XPS, TOF-SIMS and contact angle measurements. A comparison between the effectivity of ATR-photometry on chip using TE0 mode and photometry in transmission mode is discussed.
Furin proteolytically processes the heparin-binding region of extracellular superoxide dismutase
DEFF Research Database (Denmark)
Bowler, Russell P; Nicks, Mike; Olsen, Dorte Aa
2002-01-01
Extracellular superoxide dismutase (EC-SOD) is an antioxidant enzyme that attenuates brain and lung injury from oxidative stress. A polybasic region in the carboxyl terminus distinguishes EC-SOD from other superoxide dismutases and determines EC-SOD's tissue half-life and affinity for heparin....... There are two types of EC-SOD that differ based on the presence or absence of this heparin-binding region. It has recently been shown that proteolytic removal of the heparin-binding region is an intracellular event (Enghild, J. J., Thogersen, I. B., Oury, T. D., Valnickova, Z., Hojrup, P., and Crapo, J. D...... of intracellular proteases implicate furin as a processing protease. In vitro experiments using furin and purified EC-SOD suggest that furin proteolytically cleaves EC-SOD in the middle of the polybasic region and then requires an additional carboxypeptidase to remove the remaining lysines and arginines...
Mazor, Rafi; Schmid-Schönbein, Geert W
2015-01-01
Abnormal blood rheological properties seldom occur in isolation and instead are accompanied by other complications, often designated as co-morbidities. In the metabolic syndrome with complications like hypertension, diabetes and lack of normal microvascular blood flow, the underlying molecular mechanisms that simultaneously lead to elevated blood pressure and diabetes as well as abnormal microvascular rheology and other cell dysfunctions have remained largely unknown. In this review, we propose a new hypothesis for the origin of abnormal cell functions as well as multiple co-morbidities. Utilizing experimental models for the metabolic disease with diverse co-morbidities we summarize evidence for the presence of an uncontrolled extracellular proteolytic activity that causes ectodomain receptor cleavage and loss of their associated cell function. We summarize evidence for unchecked degrading proteinase activity, e.g. due to matrix metalloproteases, in patients with hypertension, Type II diabetes and obesity, in addition to evidence for receptor cleavage in the form of receptor fragments and decreased extracellular membrane expression levels. The evidence suggest that a shift in blood rheological properties and other co-morbidities may in fact be derived from a common mechanism that is due to uncontrolled proteolytic activity, i.e. an early form of autodigestion. Identification of the particular proteases involved and the mechanisms of their activation may open the door to treatment that simultaneously targets multiple co-morbidities in the metabolic syndrome.
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Elliott M McMillan
Full Text Available Hypertension is a cardiovascular disease associated with deleterious effects in skeletal and cardiac muscle. Autophagy is a degradative process essential to muscle health. Acute exercise can alter autophagic signaling. Therefore, we aimed to characterize the effects of chronic endurance exercise on autophagy in skeletal and cardiac muscle of normotensive and hypertensive rats. Male Wistar Kyoto (WKY and spontaneously hypertensive rats (SHR were assigned to a sedentary condition or 6 weeks of treadmill running. White gastrocnemius (WG of hypertensive rats had higher (p<0.05 caspase-3 and proteasome activity, as well as elevated calpain activity. In addition, skeletal muscle of hypertensive animals had elevated (p<0.05 ATG7 and LC3I protein, LAMP2 mRNA, and cathepsin activity, indicative of enhanced autophagic signaling. Interestingly, chronic exercise training increased (p<0.05 Beclin-1, LC3, and p62 mRNA as well as proteasome activity, but reduced (p<0.05 Beclin-1 and ATG7 protein, as well as decreased (p<0.05 caspase-3, calpain, and cathepsin activity. Left ventricle (LV of hypertensive rats had reduced (p<0.05 AMPKα and LC3II protein, as well as elevated (p<0.05 p-AKT, p-p70S6K, LC3I and p62 protein, which collectively suggest reduced autophagic signaling. Exercise training had little effect on autophagy-related signaling factors in LV; however, exercise training increased (p<0.05 proteasome activity but reduced (p<0.05 caspase-3 and calpain activity. Our results suggest that autophagic signaling is altered in skeletal and cardiac muscle of hypertensive animals. Regular aerobic exercise can effectively alter the proteolytic environment in both cardiac and skeletal muscle, as well as influence several autophagy-related factors in skeletal muscle of normotensive and hypertensive rats.
[Cell-derived microparticles unveil their fibrinolytic and proteolytic function].
Doeuvre, Loïc; Angles-Cano, Eduardo
2009-01-01
Cell-derived microparticles (MP) are membrane microvesicles, 0.1-1 microm in size, shed by cells following activation or during apoptosis in a variety of pathological conditions. MPs released by blood cells or by vascular endothelial cells display molecular signatures that allow their identification and functional characterization. In addition, they provide tissue factor (TF) and a procoagulant phospholipid surface. Therefore, at present, the most strongly established applied research on MPs is their procoagulant activity as a determinant of thrombotic risk in various clinical conditions. Previous studies have indicated that MPs derived from malignant cells express matrix metalloproteinases, urokinase and its receptor (uPA/uPAR) that, in the presence of plasminogen, may act in concert to degrade extracellular matrix proteins. Recently, it was shown that MPs from TNFa-stimulated endothelial cells served as a surface for interaction with plasminogen and its conversion into plasmin by the uPA/uPAR system expressed at their surface. This capacity of MPs to promote plasmin generation confers them a new profibrinolytic and proteolytic function that may be of relevance in fibrinolysis, cell migration, angiogenesis, dissemination of malignant cells, cell detachment and apoptosis.
DEFF Research Database (Denmark)
Rosenkilde, Anne Lind; Dionisio, Giuseppe; Holm, Preben Bach
2014-01-01
-terminal truncated version (HvEPB2ΔC) and a proteolytic resistant His6 tag. Maximum yield was obtained after 4 days of induction. Recombinant HvEPB2ΔC (r-HvEPB2ΔC) was purified using a single step of Ni2+-affinity chromatography. Purified protein was evaluated by SDS–PAGE, Western blotting and activity assays...... was 60 °C, thermal stability T50 value was 44 °C and the pH optimum was 4.5. r-HvEPB2ΔC was incubated with native purified barley seed storage proteins for up to 48 h. After 12 h, r-HvEPB2ΔC efficiently reduced the C and D hordeins almost completely, as evaluated by SDS–PAGE. The intensities of the B...... and γ hordein bands decreased continuously over the 48 h. No degradation occurred in the presence of E64. Recombinant hordeins (B1, B3 and γ1) were expressed in Escherichia coli. After 2 h of incubation with r-HvEPB2ΔC, an almost complete degradation of γ1 and partial digests of hordein B1 and B3 were...
Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver
2018-04-01
Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell
Hook, Vivian; Lietz, Christopher B.; Podvin, Sonia; Cajka, Tomas; Fiehn, Oliver
2018-05-01
Neuropeptides are short peptides in the range of 3-40 residues that are secreted for cell-cell communication in neuroendocrine systems. In the nervous system, neuropeptides comprise the largest group of neurotransmitters. In the endocrine system, neuropeptides function as peptide hormones to coordinate intercellular signaling among target physiological systems. The diversity of neuropeptide functions is defined by their distinct primary sequences, peptide lengths, proteolytic processing of pro-neuropeptide precursors, and covalent modifications. Global, untargeted neuropeptidomics mass spectrometry is advantageous for defining the structural features of the thousands to tens of thousands of neuropeptides present in biological systems. Defining neuropeptide structures is the basis for defining the proteolytic processing pathways that convert pro-neuropeptides into active peptides. Neuropeptidomics has revealed that processing of pro-neuropeptides occurs at paired basic residues sites, and at non-basic residue sites. Processing results in neuropeptides with known functions and generates novel peptides representing intervening peptide domains flanked by dibasic residue processing sites, identified by neuropeptidomics. While very short peptide products of 2-4 residues are predicted from pro-neuropeptide dibasic processing sites, such peptides have not been readily identified; therefore, it will be logical to utilize metabolomics to identify very short peptides with neuropeptidomics in future studies. Proteolytic processing is accompanied by covalent post-translational modifications (PTMs) of neuropeptides comprising C-terminal amidation, N-terminal pyroglutamate, disulfide bonds, phosphorylation, sulfation, acetylation, glycosylation, and others. Neuropeptidomics can define PTM features of neuropeptides. In summary, neuropeptidomics for untargeted, global analyses of neuropeptides is essential for elucidation of proteases that generate diverse neuropeptides for cell
PROTEOLYTIC PROCESSING OF VON WILLEBRAND FACTOR BY ADAMTS13 AND LEUKOCYTE PROTEASES
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Stefano Lancellotti
2013-09-01
Full Text Available ADAMTS13 is a 190 kDa zinc protease encoded by a gene located on chromosome 9q34. This protease specifically hydrolyzes von Willebrand factor (VWF multimers, thus causing VWF size reduction. ADAMTS13 belongs to the A Disintegrin And Metalloprotease with ThromboSpondin type 1 repeats (ADAMTS family, involved in proteolytic processing of many matrix proteins. ADAMTS13 consists of numerous domains including a metalloprotease domain, a disintegrin domain, several thrombospondin type 1 (TSP1 repeats, a cysteine-rich domain, a spacer domain and 2 CUB (Complement c1r/c1s, sea Urchin epidermal growth factor, and Bone morphogenetic protein domains. ADAMTS13 cleaves a single peptide bond (Tyr1605-Met1606 in the central A2 domain of the VWF molecule. This proteolytic cleavage is essential to reduce the size of ultra-large VWF polymers, which, when exposed to high shear stress in the microcirculation, are prone to form with platelets clumps, which cause severe syndromes called thrombotic microangiopathies (TMAs. In this review, we a discuss the current knowledge of structure-function aspects of ADAMTS13 and its involvement in the pathogenesis of TMAs, b address the recent findings concerning proteolytic processing of VWF multimers by different proteases, such as the leukocyte-derived serine and metallo-proteases and c indicate the direction of future investigations
DEFF Research Database (Denmark)
Skovbakke, Sarah Line; Heegaard, Peter M. H.; Larsen, Camilla J.
2015-01-01
of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release...... of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogues of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging...... flow cytometry in primary neutrophils and FPR-transfected cell lines we found that a fluorescently labelled analogue of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating...
Kemp, C M; Oliver, W T; Wheeler, T L; Chishti, A H; Koohmaraie, M
2013-07-01
Myofibrillar protein turnover is a key component of muscle growth and degeneration, requiring proteolytic enzymes to degrade the skeletal muscle proteins. The objective of this study was to investigate the role of the calpain proteolytic system in muscle growth development using μ-calpain knockout (KO) mice in comparison with control wild-type (WT) mice, and evaluate the subsequent effects of silencing this gene on other proteolytic systems. No differences in muscle development between genotypes were observed during the early stages of growth due to the up regulation of other proteolytic systems. The KO mice showed significantly greater m-calpain protein abundance (P proteolytic systems to ensure muscle protein homeostasis in vivo. Furthermore, these data contribute to the existing evidence of the importance of the calpain system's involvement in muscle growth, development, and atrophy. Collectively, these data suggest that there are opportunities to target the calpain system to promote the growth and/or restoration of skeletal muscle mass.
Intrinsic Tau Acetylation Is Coupled to Auto-Proteolytic Tau Fragmentation.
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Todd J Cohen
Full Text Available Tau proteins are abnormally aggregated in a range of neurodegenerative tauopathies including Alzheimer's disease (AD. Recently, tau has emerged as an extensively post-translationally modified protein, among which lysine acetylation is critical for normal tau function and its pathological aggregation. Here, we demonstrate that tau isoforms have different propensities to undergo lysine acetylation, with auto-acetylation occurring more prominently within the lysine-rich microtubule-binding repeats. Unexpectedly, we identified a unique intrinsic property of tau in which auto-acetylation induces proteolytic tau cleavage, thereby generating distinct N- and C-terminal tau fragments. Supporting a catalytic reaction-based mechanism, mapping and mutagenesis studies showed that tau cysteines, which are required for acetyl group transfer, are also essential for auto-proteolytic tau processing. Further mass spectrometry analysis identified the C-terminal 2nd and 4th microtubule binding repeats as potential sites of auto-cleavage. The identification of acetylation-mediated auto-proteolysis provides a new biochemical mechanism for tau self-regulation and warrants further investigation into whether auto-catalytic functions of tau are implicated in AD and other tauopathies.
Production of extracellular proteolytic enzymes by Beauveria bassiana
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Józefa Chrzanowska
2014-08-01
Full Text Available The production of proteolytic enzymes by two strains of Beauveria bassiana 278, B. bassiana 446 and one strain of Ascosphera apis 496 was analysed. It was demonstrated that the strain of B. bassiana 278 proved to be the best producer of basic and acid proteases. The influence of different environmental factors such as nitrogen and carbon sources on the production of extracellular hydrolytic enzymes was assessed. In addition the acid protease from B. bassiana was partially characterized.
CleavPredict: A Platform for Reasoning about Matrix Metalloproteinases Proteolytic Events.
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Sonu Kumar
Full Text Available CleavPredict (http://cleavpredict.sanfordburnham.org is a Web server for substrate cleavage prediction for matrix metalloproteinases (MMPs. It is intended as a computational platform aiding the scientific community in reasoning about proteolytic events. CleavPredict offers in silico prediction of cleavage sites specific for 11 human MMPs. The prediction method employs the MMP specific position weight matrices (PWMs derived from statistical analysis of high-throughput phage display experimental results. To augment the substrate cleavage prediction process, CleavPredict provides information about the structural features of potential cleavage sites that influence proteolysis. These include: secondary structure, disordered regions, transmembrane domains, and solvent accessibility. The server also provides information about subcellular location, co-localization, and co-expression of proteinase and potential substrates, along with experimentally determined positions of single nucleotide polymorphism (SNP, and posttranslational modification (PTM sites in substrates. All this information will provide the user with perspectives in reasoning about proteolytic events. CleavPredict is freely accessible, and there is no login required.
Shakerian, Mansour; Razavi, Seyed Hadi; Ziai, Seyed Ali; Khodaiyan, Faramarz; Yarmand, Mohammad Saeid; Moayedi, Ali
2015-04-01
In this study, the effects of fat (0.5 %, 3.2 % and 5.0 %), inulin (0.0 and 1.0 %) and starter culture (0.0 %, 0.5 %, 1.0 % and 1.5 %) on the angiotensin converting enzyme (ACE)-inhibitory activity of probiotic yogurt containing non-viable bacteria were assessed. Proteolytic activities of bacteria were also investigated. Yogurts were prepared either using a sole yogurt commercial culture including Streptococcus thermophilus and Lactobacillus delbrueckii subs. bulgaricus or bifidobacterium animalis BB-12 and Lactobacillus acidophilus La5 in addition to yogurt culture. Relative degrees of proteolysis were found to be considerably higher in yogurt samples than UHT milk as the control. Both regular and probiotic yogurts showed considerable ACE-inhibitory activities. Results showed that degree of proteolysis was not influenced by different fat contents, while was increased by high concentration of starter culture (1.5 % w/w) and reduced by inulin (1 % w/w). ACE-inhibitory activities of yogurt were also negatively affected by the presence of inulin and high levels of fat (5 % w/w). Moreover, yogurt containing probiotic bacteria showed higher inhibitory against ACE in comparison to the yogurt prepared with non-probiotic strains.
Bashir, Mohamed Elfatih H.; Ward, Jason M.; Cummings, Matthew; Karrar, Eltayeb E.; Root, Michael; Mohamed, Abu Bekr A.; Naclerio, Robert M.; Preuss, Daphne
2013-01-01
Background The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., “de-fatted”), and, as a result, their involvement in allergy has not been explored. Methodology/Principal Findings Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM) to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass) pollen (BGP) by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP) and endoxylanase (EXY). The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. Conclusions/Significance Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic responses is
Directory of Open Access Journals (Sweden)
Mohamed Elfatih H Bashir
Full Text Available BACKGROUND: The pollen coat is the first structure of the pollen to encounter the mucosal immune system upon inhalation. Prior characterizations of pollen allergens have focused on water-soluble, cytoplasmic proteins, but have overlooked much of the extracellular pollen coat. Due to washing with organic solvents when prepared, these pollen coat proteins are typically absent from commercial standardized allergenic extracts (i.e., "de-fatted", and, as a result, their involvement in allergy has not been explored. METHODOLOGY/PRINCIPAL FINDINGS: Using a unique approach to search for pollen allergenic proteins residing in the pollen coat, we employed transmission electron microscopy (TEM to assess the impact of organic solvents on the structural integrity of the pollen coat. TEM results indicated that de-fatting of Cynodon dactylon (Bermuda grass pollen (BGP by use of organic solvents altered the structural integrity of the pollen coat. The novel IgE-binding proteins of the BGP coat include a cysteine protease (CP and endoxylanase (EXY. The full-length cDNA that encodes the novel IgE-reactive CP was cloned from floral RNA. The EXY and CP were purified to homogeneity and tested for IgE reactivity. The CP from the BGP coat increased the permeability of human airway epithelial cells, caused a clear concentration-dependent detachment of cells, and damaged their barrier integrity. CONCLUSIONS/SIGNIFICANCE: Using an immunoproteomics approach, novel allergenic proteins of the BGP coat were identified. These proteins represent a class of novel dual-function proteins residing on the coat of the pollen grain that have IgE-binding capacity and proteolytic activity, which disrupts the integrity of the airway epithelial barrier. The identification of pollen coat allergens might explain the IgE-negative response to available skin-prick-testing proteins in patients who have positive symptoms. Further study of the role of these pollen coat proteins in allergic
Proteolytic enzymes in seawater: contribution of prokaryotes and protists
Obayashi, Y.; Suzuki, S.
2016-02-01
Proteolytic enzyme is one of the major catalysts of microbial processing of organic matter in biogeochemical cycle. Here we summarize some of our studies about proteases in seawater, including 1) distribution of protease activities in coastal and oceanic seawater, 2) responses of microbial community and protease activities in seawater to organic matter amending, and 3) possible contribution of heterotrophic protists besides prokaryotes to proteases in seawater, to clarify cleared facts and remaining questions. Activities of aminopeptidases, trypsin-type and chymotrypsin-type proteases were detected from both coastal and oceanic seawater by using MCA-substrate assay. Significant activities were detected from not only particulate (cell-associated) fraction but also dissolved fraction of seawater, especially for trypsin-type and chymotrypsin-type proteases. Hydrolytic enzymes in seawater have been commonly thought to be mainly derived from heterotrophic prokaryotes; however, it was difficult to determine actual source organisms of dissolved enzymes in natural seawater. Our experiment with addition of dissolved protein to subtropical oligotrophic Pacific water showed drastically enhancement of the protease activities especially aminopeptidases in seawater, and the prokaryotic community structure simultaneously changed to be dominant of Bacteroidetes, indicating that heterotrophic bacteria were actually one of the sources of proteases in seawater. Another microcosm experiment with free-living marine heterotrophic ciliate Paranophrys marina together with an associated bacterium showed that extracellular trypsin-type activity was mainly attributed to the ciliate. The protist seemed to work in organic matter digestion in addition to be a grazer. From the results, we propose a system of organic matter digestion by prokaryotes and protists in aquatic environments, although their actual contribution in natural environments should be estimated in future studies.
Wyss, C; Moter, A; Choi, B-K; Dewhirst, F E; Xue, Yi; Schüpbach, P; Göbel, U B; Paster, B J; Guggenheim, B
2004-07-01
So far, little phenotypic heterogeneity has been detected in cultured oral treponemes with trypsin-like proteolytic activity, and all have been assigned to the species Treponema denticola. However, comparisons of protein patterns and antigen expression in our collection of proteolytic oral treponemes occasionally identified isolates with a unique phenotype; e.g. strain OMZ 830 (=ATCC 700768), which qualified as a 'pathogen-related oral spirochaete' due to the presence of a approximately 37 kDa protein reactive with the Treponema pallidum FlaA-specific mAb H9-2. In addition to such single isolates, a homogeneous group of seven independent strains is described that were highly motile, medium-sized, proteolytic but asaccharolytic spirochaetes and were cultured from human gingivitis, periodontitis and acute necrotizing ulcerative gingivitis in medium OMIZ-Pat supplemented with 1% human serum and antibiotics. Growth of these spirochaetes in OMIZ-Pat was not dependent on, but was stimulated by, human or bovine serum. Carbohydrates were neither required nor stimulatory for growth. The protein and antigen patterns of total cell extracts of these organisms separated by SDS-PAGE were distinct from those of all previously cultured spirochaetes, with highest similarity to T. denticola. The novel spirochaete has a 2 : 4 : 2 arrangement of the periplasmic flagella, similar to T. denticola. However, the flagellin pattern as detected by immunostaining or glycan staining of Western blots readily distinguished the novel group from T. denticola. Also, distinct from reference strains of T. denticola, none of the novel isolates displayed sialidase or dentilisin activities, both of which are expressed by most strains of T. denticola. Trypsin-like activity and other enzymes as detected by API ZYM test were similar to those of T. denticola. The status of a novel species is supported by the 16S rRNA gene sequence, with 98.5% similarity to its closest cultured relative, T. denticola. The
Stanyer, Lee; Jorgensen, Wenche; Hori, Osamu; Clark, John B; Heales, Simon J R
2008-09-01
The accumulation of oxidatively modified proteins has been shown to be a characteristic feature of many neurodegenerative disorders and its regulation requires efficient proteolytic processing. One component of the mitochondrial proteolytic system is Lon, an ATP-dependent protease that has been shown to degrade oxidatively modified aconitase in vitro and may thus play a role in defending against the accumulation of oxidized matrix proteins in mitochondria. Using an assay system that allowed us to distinguish between basal and ATP-stimulated Lon protease activity, we have shown in isolated non-synaptic rat brain mitochondria that Lon protease is highly susceptible to oxidative inactivation by peroxynitrite (ONOO(-)). This susceptibility was more pronounced with regard to ATP-stimulated activity, which was inhibited by 75% in the presence of a bolus addition of 1mM ONOO(-), whereas basal unstimulated activity was inhibited by 45%. Treatment of mitochondria with a range of peroxynitrite concentrations (10-1000 microM) revealed that a decline in Lon protease activity preceded electron transport chain (ETC) dysfunction (complex I, II-III and IV) and that ATP-stimulated activity was approximately fivefold more sensitive than basal Lon protease activity. Furthermore, supplementation of mitochondrial matrix extracts with reduced glutathione, following ONOO(-) exposure, resulted in partial restoration of basal and ATP-stimulated activity, thus suggesting possible redox regulation of this enzyme complex. Taken together these findings suggest that Lon protease may be particularly vulnerable to inactivation in conditions associated with GSH depletion and elevated oxidative stress.
McSweeney, Christopher S.; Palmer, Brian; Bunch, Rowan; Krause, Denis O.
1999-01-01
Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants. Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets. Bacteria from the rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin. Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia. Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates. In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin. One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone. Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97.6%) to Clostridium botulinum NCTC 7273. Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia. This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen. In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin. PMID:10388706
Proteolytic system of blood-feeding ticks: An update on protein structures
Czech Academy of Sciences Publication Activity Database
Kovářová, Zuzana; Hobizalová, Radka; Žebrakovská, Iva; Brynda, Jiří; Řezáčová, Pavlína; Horn, Martin; Mareš, Michael
2015-01-01
Roč. 22, č. 1 (2015), s. 43-44 ISSN 1211-5894. [Discussions in Structural Molecular Biology. Annual Meeting of the Czech Society for Structural Biology /13./. 19.03.2015-21.03.2015, Nové Hrady] Institutional support: RVO:61388963 ; RVO:68378050 Keywords : blood-feeding ticks * protein structure * proteolytic system Subject RIV: CE - Biochemistry
O'Neal, Patrick; Alamdari, Nima; Smith, Ira; Poylin, Vitaliy; Menconi, Michael; Hasselgren, Per-Olof
2009-11-01
Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle-specific ubiquitin ligases atrogin-1 and MuRF1 but it is not known if atrogin-1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin-proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 microg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin-1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain-, and caspase-3-dependent protein breakdown in addition to proteasome-dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin-1 and MuRF1 mRNA levels. The same treatment increased proteasome-, cathepsin L-, and calpain-dependent proteolytic rates by approximately 40% but did not influence caspase-3-dependent proteolysis. The expression of atrogin-1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin-proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. (c) 2009 Wiley-Liss, Inc.
International Nuclear Information System (INIS)
El-Hifnawi, H.M.N.E.A.
1997-01-01
Amniotic membrane allo - and xeno grafts prepared from human foetal placenta, and their potential replacement of skin autotransplant, would significantly contribute to the success of clinical treatment of skin burns. Allo-and xenografts of human amniotic membrane should be ensured for their sterility, bio-mechanics and tissue antigenicity. The present study has been focused on sterilization and sterility assurance of the membrane grafts. Physico-chemical properties and antigenicity of the grafts await investigation. In the present study the isolation and identification of the bacteria contaminating the amniotic membrane allo-and xenografts prepared from human foetal placenta and the effect of gamma irradiation on it has been investigated. The proteolytic activity of these bacteria and the role of gamma irradiation in the control of bacterial activity were similarly investigated
Smart biomaterials: Surfaces functionalized with proteolytically stable osteoblast-adhesive peptides
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Annj Zamuner
2017-09-01
Full Text Available Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP mapped on [351–359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.
Zamuner, Annj; Brun, Paola; Scorzeto, Michele; Sica, Giuseppe; Castagliuolo, Ignazio; Dettin, Monica
2017-09-01
Engineered scaffolds for bone tissue regeneration are designed to promote cell adhesion, growth, proliferation and differentiation. Recently, covalent and selective functionalization of glass and titanium surfaces with an adhesive peptide (HVP) mapped on [351-359] sequence of human Vitronectin allowed to selectively increase osteoblast attachment and adhesion strength in in vitro assays, and to promote osseointegration in in vivo studies. For the first time to our knowledge, in this study we investigated the resistance of adhesion sequences to proteolytic digestion: HVP was completely cleaved after 5 h. In order to overcome the enzymatic degradation of the native peptide under physiological conditions we synthetized three analogues of HVP sequence. A retro-inverted peptide D-2HVP, composed of D amino acids, was completely stable in serum-containing medium. In addition, glass surfaces functionalized with D-2HVP increased human osteoblast adhesion as compared to the native peptide and maintained deposition of calcium. Interestingly, D-2HVP increased expression of IBSP, VTN and SPP1 genes as compared to HVP functionalized surfaces. Total internal reflection fluorescence microscope analysis showed cells with numerous filopodia spread on D-2HVP-functionalized surfaces. Therefore, the D-2HVP sequence is proposed as new osteoblast adhesive peptide with increased bioactivity and high proteolytic resistance.
Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications.
da Silva, Ronivaldo Rodrigues
2017-09-01
Submerged and solid-state bioprocesses have been extensively explored worldwide and employed in a number of important studies dealing with microbial cultivation for the production of enzymes. The development of these production technologies has facilitated the generation of new enzyme-based products with applications in pharmaceuticals, food, bioactive peptides, and basic research studies, among others. The applicability of microorganisms in biotechnology is potentiated because of their various advantages, including large-scale production, short time of cultivation, and ease of handling. Currently, several studies are being conducted to search for new microbial peptidases with peculiar biochemical properties for industrial applications. Bioprospecting, being an important prerequisite for research and biotechnological development, is based on exploring the microbial diversity for enzyme production. Limited information is available on the production of specific proteolytic enzymes from bacterial and fungal species, especially on the subgroups threonine and glutamic peptidases, and the seventh catalytic type, nonhydrolytic asparagine peptide lyase. This gap in information motivated the present study about these unique biocatalysts. In this study, the biochemical and biotechnological aspects of the seven catalytic types of proteolytic enzymes, namely aspartyl, cysteine, serine, metallo, glutamic, and threonine peptidase, and asparagine peptide lyase, are summarized, with an emphasis on new studies, production, catalysis, and application of these enzymes.
Pranjol, Md Zahidul I; Gutowski, Nicholas J; Hannemann, Michael; Whatmore, Jacqueline L
2018-01-01
Epithelial ovarian cancer (EOC) frequently metastasises to the omentum, a process that requires pro-angiogenic activation of human omental microvascular endothelial cells (HOMECs) by tumour-secreted factors. We have previously shown that ovarian cancer cells secrete a range of factors that induce pro-angiogenic responses e.g. migration, in HOMECs including the lysosomal protease cathepsin D (CathD). However, the cellular mechanism by which CathD induces these cellular responses is not understood. The aim of this study was to further examine the pro-angiogenic effects of CathD in HOMECs i.e. proliferation and migration, to investigate whether these effects are dependent on CathD catalytic activity and to delineate the intracellular signalling kinases activated by CathD. We report, for the first time, that CathD significantly increases HOMEC proliferation and migration via a non-proteolytic mechanism resulting in activation of ERK1/2 and AKT. These data suggest that EOC cancer secreted CathD acts as an extracellular ligand and may play an important pro-angiogenic, and thus pro-metastatic, role by activating the omental microvasculature during EOC metastasis to the omentum. Copyright © 2017 Elsevier B.V. All rights reserved.
Settachaimongkon, Sarn; Nout, M J Robert; Antunes Fernandes, Elsa C; Hettinga, Kasper A; Vervoort, Jacques M; van Hooijdonk, Toon C M; Zwietering, Marcel H; Smid, Eddy J; van Valenberg, Hein J F
2014-05-02
Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L. delbrueckii subsp. bulgaricus was investigated in terms of microbial growth, acidification and changes in the biochemical composition of milk during set-yoghurt fermentation. A complementary metabolomics approach was applied for global characterization of volatile and non-volatile polar metabolite profiles of yoghurt associated with proteolytic activity of the individual strains in the starter cultures. The results demonstrated that only non-proteolytic S. thermophilus (Prt-) strain performed proto-cooperation with L. delbrueckii subsp. bulgaricus. The proto-cooperation resulted in significant higher populations of the two species, faster milk acidification, significant abundance of aroma volatiles and non-volatile metabolites desirable for a good organoleptic quality of yoghurt. Headspace SPME-GC/MS and (1)H NMR resulted in the identification of 35 volatiles and 43 non-volatile polar metabolites, respectively. Furthermore, multivariate statistical analysis allows discriminating set-yoghurts fermented by different types of starter cultures according to their metabolite profiles. Our finding underlines that selection of suitable strain combinations in yoghurt starters is important for achieving the best technological performance regarding the quality of product. Copyright © 2014 Elsevier B.V. All rights reserved.
Genetics of Proteolytic Enzymes of Lactococci and Their Role in Cheese Flavor Development
Kok, Jan
In recent years, knowledge of the genetics and biochemistry of the enzymes that constitute the proteolytic system of starter lactococci has increased tremendously. This paper summarizes the data obtained largely in the last 5 yr of intensive research by various research groups operative in this
Nhan, Hoang S; Chiang, Karen; Koo, Edward H
2015-01-01
The amyloid precursor protein (APP) has occupied a central position in Alzheimer's disease (AD) pathophysiology, in large part due to the seminal role of amyloid-β peptide (Aβ), a proteolytic fragment derived from APP. Although the contribution of Aβ to AD pathogenesis is accepted by many in the research community, recent studies have unveiled a more complicated picture of APP's involvement in neurodegeneration in that other APP-derived fragments have been shown to exert pathological influences on neuronal function. However, not all APP-derived peptides are neurotoxic, and some even harbor neuroprotective effects. In this review, we will explore this complex picture by first discussing the pleiotropic effects of the major APP-derived peptides cleaved by multiple proteases, including soluble APP peptides (sAPPα, sAPPβ), various C- and N-terminal fragments, p3, and APP intracellular domain fragments. In addition, we will highlight two interesting sequences within APP that likely contribute to this duality in APP function. First, it has been found that caspase-mediated cleavage of APP in the cytosolic region may release a cytotoxic peptide, C31, which plays a role in synapse loss and neuronal death. Second, recent studies have implicated the -YENPTY- motif in the cytoplasmic region as a domain that modulates several APP activities through phosphorylation and dephosphorylation of the first tyrosine residue. Thus, this review summarizes the current understanding of various APP proteolytic products and the interplay among them to gain deeper insights into the possible mechanisms underlying neurodegeneration and AD pathophysiology.
International Nuclear Information System (INIS)
Beausoleil, Michel S; Schulze, Erika B; Goodale, David; Postenka, Carl O; Allan, Alison L
2011-01-01
Osteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other. To study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior. All three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells. The results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer
Directory of Open Access Journals (Sweden)
Yamara A. S. de Menezes
2014-03-01
Full Text Available Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L. Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogenolytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.
Enzymatic surface hydrolysis of polyamide 6,6 with mixtures of proteolytic and lipolytic enzymes.
Parvinzadeh Gashti, Mazeyar; Assefipour, Reza; Kiumarsi, Amir; Parvinzadeh Gashti, Mahyar
2013-01-01
This study investigated the changes induced on nylon 6,6 fabric by a mixture of proteolytic and lipolytic enzymes. Technical measurements were studied including those of Fourier-transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), weight loss (WL), bending lengths (BL), scanning electron microscopy (SEM), moisture absorbency (MA), and reflectance spectroscopy (RS). For this purpose, nylon 6,6 fabrics were treated separately with different concentrations of protease and lipase mixtures in solution. The dyeing process was then carried out on the treated fabrics with two reactive and acid dyes. The intensity of major peaks in the FTIR spectra of the protease-treated samples is in favor of chemical changes the polypeptide functional groups in the fabrics. Thermal studies also show a significant decrease in the thermal degradation temperature of the treated polymer at temperatures higher than 400°C. The protease and lipase mixtures decreased the sample weight, while lipase intensified the weight loss comparing with protease. It was observed that the concentration of lipase enzyme had a direct influence on the darkness of dyed samples.
[Protease activity of microflora in the oral cavity of patients with periodontitis].
Voropaeva, E A; Baĭrakova, A L; Bichucher, A M; D'iakov, V L; Kozlov, L V
2008-01-01
Microbial spectrum and non-specific as well as specific IgA1 protease activity of isolated microorganisms were investigated in gingival liquid of patients with periodontitis. Microorganisms from the gingival liqud of these patients belonged to conditional-pathogenic obligate and facultatively anaerobic bacteria. 24 strains of microorganisms have been identified. Nonspecific proteolytic activity was found in the following microorganisms: Actinomyces israelii, Actinomyces naeslundii, Aerococcus viridans, Bifidobacterium longum, Neisseria subflave, Streptococcus parvulus, Eubacterium alactolyticum, Lactobaccilus catenoforme, Bacillus spp. Specific IgA1-protease activity and lack of proteolytic activity towards IgG was found in Streptococcus acidominimus, Streptococcus hansenii, Streptococcus salivarius, Leptotrychia buccalis, Staphylococcus haemolyticus and Neisseria sicca. No proteolytic activity was found in cultivation medium of Eubacterium alactolyticum (1 strain), Prevotella buccalis, Aerococcus viridans and Streptococcus sanguis.
International Nuclear Information System (INIS)
Nishitani, Koji; Namba, Masayoshi; Ohkubo, Shigeki; Kimoto, Tetsuo
1985-01-01
Production of proteolytic enzymes by human fibroblasts in the process of transformation was investigated. The cells used were normal human fibroblasts (KSM-6) and their in vitro counterparts transformed by treatment with 60 Co gamma rays (KMST-6). Cells seeded by treatment with EDTA were cultured in a serum free medium. Proteolytic enzymes in the culture medium of cells were assayed using a synthetic substrate, N-α-(p-tosyl)-L-arginine ( 3 H) methyl ester hydrochloride. The transformed cells (KMST-6) produced a larger amount of enzymes than normal cells (KMS-6). The enzyme production in both cell lines was high in the exponential growth stage and then decreased as the cells reached confluency. The proteolytic enzymes produced by these cells were trypsin- and thrombin-like enzymes. Cell growth of KMST-6 or KMS-6 was not inhibited by the addition of protease inhibitors to the culture medium. (author)
Shtenberg, Giorgi; Massad-Ivanir, Naama; Moscovitz, Oren; Engin, Sinem; Sharon, Michal; Fruk, Ljiljana; Segal, Ester
2013-02-05
A multifunctional porous Si biosensor that can both monitor the enzymatic activity of minute samples and allow subsequent retrieval of the entrapped proteolytic products for mass spectrometry analysis is described. The biosensor is constructed by DNA-directed/reversible immobilization of enzymes onto a Fabry-Pérot thin film. We demonstrate high enzymatic activity levels of the immobilized enzymes (more than 80%), while maintaining their specificity. Mild dehybridization conditions allow enzyme recycling and facile surface regeneration for consecutive biosensing analysis. The catalytic activity of the immobilized enzymes is monitored in real time by reflective interferometric Fourier transform spectroscopy. The real-time analysis of minute quantities of enzymes (concentrations at least 1 order of magnitude lower, 0.1 mg mL(-1), in comparison to previous reports, 1 mg mL(-1)), in particular proteases, paves the way for substrate profiling and the identification of cleavage sites. The biosensor configuration is compatible with common proteomic methods and allows for a successful downstream mass spectrometry analysis of the reaction products.
López-Jiménez, Alberto J; Basak, Trayambak; Vanacore, Roberto M
2017-10-13
Lysyl oxidase-like-2 (LOXL2) is an enzyme secreted into the extracellular matrix that crosslinks collagens by mediating oxidative deamination of lysine residues. Our previous work demonstrated that this enzyme crosslinks the 7S domain, a structural domain that stabilizes collagen IV scaffolds in the basement membrane. Despite its relevant role in extracellular matrix biosynthesis, little is known about the structural requirements of LOXL2 that enable collagen IV crosslinking. In this study, we demonstrate that LOXL2 is processed extracellularly by serine proteases, generating a 65-kDa form lacking the first two scavenger receptor cysteine-rich domains. Site-specific mutagenesis to prevent proteolytic processing generated a full-length enzyme that is active in vitro toward a soluble substrate, but fails to crosslink insoluble collagen IV within the extracellular matrix. In contrast, the processed form of LOXL2 binds to collagen IV and crosslinks the 7S domain. Together, our data demonstrate that proteolytic processing is an important event that allows LOXL2-mediated crosslinking of basement membrane collagen IV. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Kanai, Stanley M; Edwards, Alethia J; Rurik, Joel G; Osei-Owusu, Patrick; Blumer, Kendall J
2017-11-24
Regulator of G protein signaling 2 (RGS2) controls signaling by receptors coupled to the G q/11 class heterotrimeric G proteins. RGS2 deficiency causes several phenotypes in mice and occurs in several diseases, including hypertension in which a proteolytically unstable RGS2 mutant has been reported. However, the mechanisms and functions of RGS2 proteolysis remain poorly understood. Here we addressed these questions by identifying degradation signals in RGS2, and studying dynamic regulation of G q/11 -evoked Ca 2+ signaling and vascular contraction. We identified a novel bipartite degradation signal in the N-terminal domain of RGS2. Mutations disrupting this signal blunted proteolytic degradation downstream of E3 ubiquitin ligase binding to RGS2. Analysis of RGS2 mutants proteolyzed at various rates and the effects of proteasome inhibition indicated that proteolytic degradation controls agonist efficacy by setting RGS2 protein expression levels, and affecting the rate at which cells regain agonist responsiveness as synthesis of RGS2 stops. Analyzing contraction of mesenteric resistance arteries supported the biological relevance of this mechanism. Because RGS2 mRNA expression often is strikingly and transiently up-regulated and then down-regulated upon cell stimulation, our findings indicate that proteolytic degradation tightly couples RGS2 transcription, protein levels, and function. Together these mechanisms provide tight temporal control of G q/11 -coupled receptor signaling in the cardiovascular, immune, and nervous systems. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
Katchman, Alexander; Yang, Lin; Zakharov, Sergey I; Kushner, Jared; Abrams, Jeffrey; Chen, Bi-Xing; Liu, Guoxia; Pitt, Geoffrey S; Colecraft, Henry M; Marx, Steven O
2017-08-22
Calcium influx through the voltage-dependent L-type calcium channel (Ca V 1.2) rapidly increases in the heart during "fight or flight" through activation of the β-adrenergic and protein kinase A (PKA) signaling pathway. The precise molecular mechanisms of β-adrenergic activation of cardiac Ca V 1.2, however, are incompletely known, but are presumed to require phosphorylation of residues in α 1C and C-terminal proteolytic cleavage of the α 1C subunit. We generated transgenic mice expressing an α 1C with alanine substitutions of all conserved serine or threonine, which is predicted to be a potential PKA phosphorylation site by at least one prediction tool, while sparing the residues previously shown to be phosphorylated but shown individually not to be required for β-adrenergic regulation of Ca V 1.2 current (17-mutant). A second line included these 17 putative sites plus the five previously identified phosphoregulatory sites (22-mutant), thus allowing us to query whether regulation requires their contribution in combination. We determined that acute β-adrenergic regulation does not require any combination of potential PKA phosphorylation sites conserved in human, guinea pig, rabbit, rat, and mouse α 1C subunits. We separately generated transgenic mice with inducible expression of proteolytic-resistant α 1C Prevention of C-terminal cleavage did not alter β-adrenergic stimulation of Ca V 1.2 in the heart. These studies definitively rule out a role for all conserved consensus PKA phosphorylation sites in α 1C in β-adrenergic stimulation of Ca V 1.2, and show that phosphoregulatory sites on α 1C are not redundant and do not each fractionally contribute to the net stimulatory effect of β-adrenergic stimulation. Further, proteolytic cleavage of α 1C is not required for β-adrenergic stimulation of Ca V 1.2.
Active learners in sustainable electronics and it
DEFF Research Database (Denmark)
Schultz, Ole
This poster-presentation is about active learning in a course sustainable wireless electronics and it. Active learning understood as practical lab-exercises and a team chosen project.......This poster-presentation is about active learning in a course sustainable wireless electronics and it. Active learning understood as practical lab-exercises and a team chosen project....
Fibrin Clots Are Equilibrium Polymers That Can Be Remodeled Without Proteolytic Digestion
Chernysh, Irina N.; Nagaswami, Chandrasekaran; Purohit, Prashant K.; Weisel, John W.
2012-01-01
Fibrin polymerization is a necessary part of hemostasis but clots can obstruct blood vessels and cause heart attacks and strokes. The polymerization reactions are specific and controlled, involving strong knob-into-hole interactions to convert soluble fibrinogen into insoluble fibrin. It has long been assumed that clots and thrombi are stable structures until proteolytic digestion. On the contrary, using the technique of fluorescence recovery after photobleaching, we demonstrate here that the...
Directory of Open Access Journals (Sweden)
Andrew T N Tebbenkamp
Full Text Available N-terminal fragments of mutant huntingtin (htt that terminate between residues 90-115, termed cleavage product A or 1 (cp-A/1, form intracellular and intranuclear inclusion bodies in the brains of patients with Huntington's disease (HD. These fragments appear to be proteolytic products of the full-length protein. Here, we use an HEK293 cell culture model to investigate huntingtin proteolytic processing; previous studies of these cells have demonstrated cleavage of htt to cp-A/1 like htt fragments.Recombinant N-terminal htt fragments, terminating at residue 171 (also referred to as cp-B/2 like, were efficiently cleaved to produce cp-A/1 whereas fragments representing endogenous caspase, calpain, and metalloproteinase cleavage products, terminating between residues 400-600, were inefficiently cleaved. Using cysteine-labeling techniques and antibody binding mapping, we localized the C-terminus of the cp-A/1 fragments produced by HEK293 cells to sequences minimally limited by cysteine 105 and an antibody epitope composed of residues 115-124. A combination of genetic and pharmacologic approaches to inhibit potential proteases, including γ-secretase and calpain, proved ineffective in preventing production of cp-A/1.Our findings indicate that HEK293 cells express a protease that is capable of efficiently cleaving cp-B/2 like fragments of htt with normal or expanded glutamine repeats. For reasons that remain unclear, this protease cleaves longer htt fragments, with normal or expanded glutamine expansions, much less efficiently. The protease in HEK293 cells that is capable of generating a cp-A/1 like htt fragment may be a novel protease with a high preference for a cp-B/2-like htt fragment as substrate.
Energy Technology Data Exchange (ETDEWEB)
Han, Myung-Geun, E-mail: mghan@bnl.gov [Condensed Matter Physics & Materials Science, Brookhaven National Laboratory, Upton, NY 11973 (United States); Garlow, Joseph A. [Condensed Matter Physics & Materials Science, Brookhaven National Laboratory, Upton, NY 11973 (United States); Materials Science and Engineering Department, Stony Brook University, Stony Brook, NY 11794 (United States); Marshall, Matthew S.J.; Tiano, Amanda L. [Department of Chemistry, Stony Brook University, Stony Brook, NY 11974 (United States); Wong, Stanislaus S. [Condensed Matter Physics & Materials Science, Brookhaven National Laboratory, Upton, NY 11973 (United States); Department of Chemistry, Stony Brook University, Stony Brook, NY 11974 (United States); Cheong, Sang-Wook [Department of Physics and Astronomy, Rutgers Center for Emergent Materials, Rutgers University, Piscataway, NJ 08854 (United States); Walker, Frederick J.; Ahn, Charles H. [Department of Applied Physics and Center for Research on Interface Structures and Phenomena, Yale University, New Haven, CT 06520 (United States); Department of Mechanical Engineering and Materials Science, Yale University, New Haven, CT 06520 (United States); Zhu, Yimei [Condensed Matter Physics & Materials Science, Brookhaven National Laboratory, Upton, NY 11973 (United States)
2017-05-15
Highlights: • Electron-beam-induced-current (EBIC) and active secondary-electron voltage-contrast (SE-VC) are demonstrated in STEM mode combined with in situ electrical biasing in a TEM. • Electrostatic potential maps in ferroelectric thin films, multiferroic nanowires, and single crystals obtained by off-axis electron holography were compared with EBIC and SE-VC data. • Simultaneous EBIC and active SE-VC performed with atomic resolution STEM are demonstrated. - Abstract: The ability to map out electrostatic potentials in materials is critical for the development and the design of nanoscale electronic and spintronic devices in modern industry. Electron holography has been an important tool for revealing electric and magnetic field distributions in microelectronics and magnetic-based memory devices, however, its utility is hindered by several practical constraints, such as charging artifacts and limitations in sensitivity and in field of view. In this article, we report electron-beam-induced-current (EBIC) and secondary-electron voltage-contrast (SE-VC) with an aberration-corrected electron probe in a transmission electron microscope (TEM), as complementary techniques to electron holography, to measure electric fields and surface potentials, respectively. These two techniques were applied to ferroelectric thin films, multiferroic nanowires, and single crystals. Electrostatic potential maps obtained by off-axis electron holography were compared with EBIC and SE-VC to show that these techniques can be used as a complementary approach to validate quantitative results obtained from electron holography analysis.
Mass spectrometry analysis of proteome-wide proteolytic post-translational degradation of proteins
Shen, Yufeng; Hixson, Kim K.; Tolić, Nikola; Camp, David G.; Purvine, Samuel O.; Moore, Ronald J.; Smith, Richard D.
2008-01-01
Protein proteolytic degradation is an essential component to proper cell function and its life cycle. Here, we study the protein degradation in yeast Saccharomyces cerevisiae cells on a proteome-wide scale by detection of the intermediate peptides produced from the intracellular degradation of proteins using sequencing-based tandem mass spectrometry. By tracing the detected ~1,100 peptides and their ~200 protein substrate origins we obtain evidence for new insights into the proteome-wide prot...
Extracellular proteolytic enzymes produced by human pathogenic Vibrio species
Directory of Open Access Journals (Sweden)
Shin-Ichi eMiyoshi
2013-11-01
Full Text Available Bacteria in the genus Vibrio produce extracellular proteolytic enzymes to obtain nutrients via digestion of various protein substrates. However, the enzymes secreted by human pathogenic species have been documented to modulate the bacterial virulence. Several species including Vibrio cholerae and V. vulnificus are known to produce thermolysin-like metalloproteases termed vibriolysin. The vibriolysin from V. vulnificus, a causative agent of serious systemic infection, is a major toxic factor eliciting the secondary skin damage characterized by formation of the hemorrhagic brae. The vibriolysin from intestinal pathogens may play indirect roles in pathogenicity because it can activate protein toxins and hemagglutinin by the limited proteolysis and can affect the bacterial attachment to or detachment from the intestinal surface by degradation of the mucus layer. Two species causing wound infections, V. alginolyticus and V. parahaemolyticus, produce another metalloproteases so-called collagenases. Although the detailed pathological roles have not been studied, the collagenase is potent to accelerate the bacterial dissemination through digestion of the protein components of the extracellular matrix. Some species produce cymotrypsin-like serine proteases, which may also affect the bacterial virulence potential. The intestinal pathogens produce sufficient amounts of the metalloprotease at the small intestinal temperature; however, the metalloprotease production by extra-intestinal pathogens is much higher around the body surface temperature. On the other hand, the serine protease is expressed only in the absence of the metalloprotease.
Directory of Open Access Journals (Sweden)
Mirian Pereira Rodarte
2011-07-01
Full Text Available One hundred forty-four microorganisms previously isolated from coffee fruit (Coffea arabica were grown on casein agar to evaluate their proteolytic activities. Fifty percent of filamentous fungi, 52.5% of bacteria and 2.6% of yeasts were able to secrete proteases. Positiveisolates were further examined in liquid culture for their protease activities by hydrolysis of casein at different pH values (5.0, 7.0 and 9.0 at 30 oC. Bacillus megaterium, B. subtilis, Enterobacteragglomerans, Kurthia sp, Pseudomonas paucimobilis and Tatumella ptyseos demonstrated the highest proteolytic activities at pH 9.0. One yeast isolate, Citeromyces matritensis, had a proteolytic activityof 2.40 U at pH 5.0. Aspergillus dimorphicus, A. ochraceus, Fusarium moniliforme, F. solani, Penicillium fellutanum and P. waksmanii showed the highest activities. Of the bacterial isolates, the highestenzyme activities were observed in B. subtilis 333 (27.1 U, Tatumella ptyseos (27.0 U and B. megaterium 817 (26.2 U. Of the filamentous fungi, Aspergillus ochraceus (48.7 U, Fusarium moniliforme 221 (37.5 U and F. solani 359 (37.4 U had the highest activities at pH 9.0. Este trabalho teve por objetivos avaliar a capacidade de secreção de proteases extracelulares por 144 microrganismos, previamente isoladosde grãos de café (Coffea arabica durante fermentação por via seca, e determinar a atividade das enzimas produzidas. Os microrganismos foram cultivados em ágar-caseína para avaliação da produção de enzimas proteolíticas. Dos 40 isolados de bactéria presentes na amostra, 52,5% apresentaram resultado positivo para o teste. Considerando os 66 isolados de fungos filamentosos, 50% foram capazes de secretar proteases, enquanto que dos 38 isolados de leveduras, apenas 2,6% conseguiram promover a hidrólise da caseína do meio. Os isolados que apresentaram capacidade de secreção de proteases foram, posteriormente, cultivados em meio líquido para a determinação da atividade
Murugan, Ravichandran N; Jacob, Binu; Ahn, Mija; Hwang, Eunha; Sohn, Hoik; Park, Hyo-Nam; Lee, Eunjung; Seo, Ji-Hyung; Cheong, Chaejoon; Nam, Ky-Youb; Hyun, Jae-Kyung; Jeong, Ki-Woong; Kim, Yangmee; Shin, Song Yub; Bang, Jeong Kyu
2013-01-01
Much attention has been focused on the design and synthesis of potent, cationic antimicrobial peptides (AMPs) that possess both antimicrobial and anti-inflammatory activities. However, their development into therapeutic agents has been limited mainly due to their large size (12 to 50 residues in length) and poor protease stability. In an attempt to overcome the issues described above, a set of ultra-short, His-derived antimicrobial peptides (HDAMPs) has been developed for the first time. Through systematic tuning of pendant hydrophobic alkyl tails at the N(π)- and N(τ)-positions on His, and the positive charge of Arg, much higher prokaryotic selectivity was achieved, compared to human AMP LL-37. Additionally, the most potent HDAMPs showed promising dual antimicrobial and anti-inflammatory activities, as well as anti-methicillin-resistant Staphylococcus aureus (MRSA) activity and proteolytic resistance. Our results from transmission electron microscopy, membrane depolarization, confocal laser-scanning microscopy, and calcein-dye leakage experiments propose that HDAMP-1 kills microbial cells via dissipation of the membrane potential by forming pore/ion channels on bacterial cell membranes. The combination of the ultra-short size, high-prokaryotic selectivity, potent anti-MRSA activity, anti-inflammatory activity, and proteolytic resistance of the designed HDAMP-1, -3, -5, and -6 makes these molecules promising candidates for future antimicrobial therapeutics.
Robinson, Paulette M; Smith, Tyler S; Patel, Dilan; Dave, Meera; Lewin, Alfred S; Pi, Liya; Scott, Edward W; Tuli, Sonal S; Schultz, Gregory S
2012-12-13
Connective tissue growth factor (CTGF) is a fibrogenic cytokine that is up-regulated by TGF-β and mediates most key fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. This study addresses the role of proteolytic processing of CTGF in human corneal fibroblasts (HCF) stimulated with TGF-β, normal ocular tissues and wounded corneas. Proteolytic processing of CTGF in HCF cultures, normal animal eyes, and excimer laser wounded rat corneas were examined by Western blot. The identity of a 21-kDa band was determined by tandem mass spectrometry, and possible alternative splice variants of CTGF were assessed by 5' Rapid Amplification of cDNA Ends (RACE). HCF stimulated by TGF-β contained full length 38-kDa CTGF and fragments of 25, 21, 18, and 13 kDa, while conditioned medium contained full length 38- and a 21-kDa fragment of CTGF that contained the middle "hinge" region of CTGF. Fragmentation of recombinant CTGF incubated in HCF extracts was blocked by the aspartate protease inhibitor, pepstatin. Normal mouse, rat, and rabbit whole eyes and rabbit ocular tissues contained abundant amounts of C-terminal 25- and 21-kDa fragments and trace amounts of 38-kDa CTGF, although no alternative transcripts were detected. All forms of CTGF (38, 25, and 21 kDa) were detected during healing of excimer ablated rat corneas, peaking on day 11. Proteolytic processing of 38-kDa CTGF occurs during corneal wound healing, which may have important implications in regulation of corneal scar formation.
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
Institute of Scientific and Technical Information of China (English)
XIEXiao-mei; LIAOMin; LIUWei-ping; SusanneKLOSE
2004-01-01
Combined effects on soil microbial activity of nutrient and pesticide management in hybrid rice double annual cropping system were studied. Results of field experiment demonstrated significant changes in soil microbial biomass phospholipid contents,abundance of heterotrophic bacteria and proteolytic bacteria, electron transport system (ETS)/dehydrogenase activity, soil protein contents under different management practices and at various growth stages. Marked depletions in the soil microbial biomass phospholipid contents were found with the advancement of crop growth stages, while the incorporation of fertilizers and/or pesticides also induced slight changes, and the lowest microbial biomass phospholipid content was found with pesticides application alone. A decline in the bacterial abundance of heterotrophic bacteria and proteolytic bacteria was observed during the continuance of crop growth, while the lowest abundance of heterotrophic bacteria and proteolyrJc bacteria was found with pesticides application alone, which coincided with the decline of soil microbial biomass. A consistent increase in the electron transport svstem activit), was measured during the different crop growth stages of rice. The use of fertilizers (NPK) alone or combined with pesticides increased it, while a decline was noticed with pesticides application alone as compared with the control.The soil protein content was found to be relatively stable with fertilizers and/or pesticides application at various growth stages in both crops undertaken, but notable changes were detected at different growrh stages
Effects of the electron-phonon coupling activation in collision cascades
Energy Technology Data Exchange (ETDEWEB)
Zarkadoula, Eva, E-mail: zarkadoulae@ornl.gov [Materials Science & Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (United States); Samolyuk, German [Materials Science & Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (United States); Weber, William J. [Materials Science & Technology Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831 (United States); Department of Materials Science & Engineering, University of Tennessee, Knoxville, TN 37996 (United States)
2017-07-15
Using the two-temperature (2T-MD) model in molecular dynamics simulations, we investigate the condition of switching the electronic stopping term off when the electron-phonon coupling is activated in the damage production due to 50 keV Ni ion cascades in Ni and equiatomic NiFe. Additionally, we investigate the effect of the electron-phonon coupling activation time in the damage production. We find that the switching condition has negligible effect in the produced damage, while the choice of the activation time of the electron-phonon coupling can affect the amount of surviving damage. - Highlights: •The electron-phonon interactions in irradiation affect the energy dissipation. •The resulting damage depends on the electron-phonon interaction activation time. •The electronic stopping acts on the ions before the electron-phonon interactions.
Sensitive microplate assay for the detection of proteolytic enzymes using radiolabeled gelatin
International Nuclear Information System (INIS)
Robertson, B.D.; Kwan-Lim, G.E.; Maizels, R.M.
1988-01-01
A sensitive, microplate assay is described for the detection of a wide range of proteolytic enzymes, using radio-iodine-labeled gelatin as substrate. The technique uses the Bolton-Hunter reagent to label the substrate, which is then coated onto the wells of polyvinyl chloride microtiter plates. By measuring the radioactivity released the assay is able to detect elastase, trypsin, and collagenase in concentrations of 1 ng/ml or less, while the microtiter format permits multiple sample handling and minimizes sample volumes required for analysis
DEFF Research Database (Denmark)
Almholt, Kasper; Hebsgaard, Josephine B; Nansen, Anneline
2018-01-01
the potential in mice of an Ab that blocks the proteolytic capacity of uPA in the CIA model and the delayed-type hypersensitivity arthritis model. A second aim was to determine the cellular origins of uPA and the uPA receptor (uPAR) in joint tissue from patients with rheumatoid arthritis. A mAb that neutralizes...
Low Proteolytic Clipping of Histone H3 in Cervical Cancer
Sandoval-Basilio, Jorge; Serafín-Higuera, Nicolás; Reyes-Hernandez, Octavio D.; Serafín-Higuera, Idanya; Leija-Montoya, Gabriela; Blanco-Morales, Magali; Sierra-Martínez, Monica; Ramos-Mondragon, Roberto; García, Silvia; López-Hernández, Luz Berenice; Yocupicio-Monroy, Martha; Alcaraz-Estrada, Sofia L.
2016-01-01
Chromatin in cervical cancer (CC) undergoes chemical and structural changes that alter the expression pattern of genes. Recently, a potential mechanism, which regulates gene expression at transcriptional levels is the proteolytic clipping of histone H3. However, until now this process in CC has not been reported. Using HeLa cells as a model of CC and human samples from patients with CC, we identify that the H3 cleavage was lower in CC compared with control tissue. Additionally, the histone H3 clipping was performed by serine and aspartyl proteases in HeLa cells. These results suggest that histone H3 clipping operates as part of post-translational modification system in CC. PMID:27698925
Skovbakke, Sarah Line; Heegaard, Peter M H; Larsen, Camilla J; Franzyk, Henrik; Forsman, Huamei; Dahlgren, Claes
2015-01-15
Immunomodulatory host defense peptides (HDPs) are considered to be lead compounds for novel anti-sepsis and anti-inflammatory agents. However, development of drugs based on HDPs has been hampered by problems with toxicity and low bioavailability due to in vivo proteolysis. Here, a subclass of proteolytically stable HDP mimics consisting of lipidated α-peptide/β-peptoid oligomers was investigated for their effect on neutrophil function. The most promising compound, Pam-(Lys-βNSpe)6-NH2, was shown to inhibit formyl peptide receptor 2 (FPR2) agonist-induced neutrophil granule mobilization and release of reactive oxygen species. The potency of Pam-(Lys-βNSpe)6-NH2 was comparable to that of PBP10, the most potent FPR2-selective inhibitor known. The immunomodulatory effects of structural analogs of Pam-(Lys-βNSpe)6-NH2 emphasized the importance of both the lipid and peptidomimetic parts. By using imaging flow cytometry in primary neutrophils and FPR-transfected cell lines, we found that a fluorescently labeled analog of Pam-(Lys-βNSpe)6-NH2 interacted selectively with FPR2. Furthermore, the interaction between Pam-(Lys-βNSpe)6-NH2 and FPR2 was found to prevent binding of the FPR2-specific activating peptide agonist Cy5-WKYMWM, while the binding of an FPR1-selective agonist was not inhibited. To our knowledge, Pam-(Lys-βNSpe)6-NH2 is the first HDP mimic found to inhibit activation of human neutrophils via direct interaction with FPR2. Hence, we consider Pam-(Lys-βNSpe)6-NH2 to be a convenient tool in the further dissection of the role of FPR2 in inflammation and homeostasis as well as for investigation of the importance of neutrophil stimulation in anti-infective therapy involving HDPs. Copyright © 2014 Elsevier Inc. All rights reserved.
Peng, Tao; Huang, Bingzhen; Sun, Yao; Lu, Yongbo; Bonewald, Lynda; Chen, Shuo; Butler, William T; Feng, Jerry Q; D'Souza, Rena N; Qin, Chunlin
2009-01-01
Dentin matrix protein 1 (DMP1) is present in the extracellular matrix (ECM) of dentin and bone as processed NH(2)- and COOH-terminal fragments, resulting from proteolytic cleavage at the NH(2) termini of 4 aspartic acid residues during rat DMP1 processing. One cleavage site residue, Asp(181) (corresponding to Asp(197) of mouse DMP1), and its flanking region are highly conserved across species. We speculate that cleavage at the NH(2) terminus of Asp(197) of mouse DMP1 represents an initial, first-step scission in the whole cascade of proteolytic processing. To test if Asp(197) is critical for initiating the proteolytic processing of mouse DMP1, we substituted Asp(197) with Ala(197) by mutating the corresponding nucleotides of mouse cDNA that encode this amino acid residue. This mutant DMP1 cDNA was cloned into a pcDNA3.1 vector. Data from transfection experiments indicated that this single substitution blocked the proteolytic processing of mouse DMP1 in HEK-293 cells, indicating that cleavage at the NH(2) terminus of Asp(197) is essential for exposing other cleavage sites for the conversion of DMP1 to its fragments. The NH(2)-terminal fragment of DMP1 occurs as a proteoglycan form (DMP1-PG) that contains a glycosaminoglycan (GAG) chain. Previously, we showed that a GAG chain is linked to Ser(74) in rat DMP1 (Ser(89) in mouse DMP1). To confirm that mouse DMP1-PG possesses a single GAG chain attached to Ser(89), we substituted Ser(89) by Gly(89). Data from transfection analysis indicated that this substitution completely prevented formation of the GAG-containing form, confirming that DMP1-PG contains a single GAG chain attached to Ser(89) in mouse DMP1. Copyright 2008 S. Karger AG, Basel.
Golden, Max C; Wanless, Brandon J; David, Jairus R D; Kottapalli, Bala; Lineback, D Scott; Talley, Ryan J; Glass, Kathleen A
2017-08-01
Clostridium botulinum may be of concern in prepared refrigerated meals, for which strict cold chain management cannot be guaranteed. This study evaluated the effect of temperature, product composition, and cultured celery juice powder (CCJP) as a source of nitrite on the inhibition of botulinum toxin formation in two experimental (meat- and vegetable-based) prepared meals. Data obtained from the challenge study were compared with a published mathematical model to determine whether the model is fail-safe with regard to the tested meals. Treatments were inoculated with proteolytic C. botulinum, vacuum packaged, cooked at 90°C for 10 min, and assayed for botulinum toxin at appropriate intervals in samples stored at 10, 15, or 20°C for up to 8 weeks. None of the treatments stored at 10°C for 8 weeks supported toxin production by proteolytic C. botulinum. The addition of CCJP delayed toxin production by 1 and 3 weeks in cauliflower potatoes and in Dijon pork, respectively, stored at 15°C. Toxin production was delayed by 1 week at 20°C when CCJP was added to the cauliflower potatoes. This study found that the predictive model was fail-safe but was overly conservative for the experimental meals described. Finally, this study confirms that product composition, the addition of nitrite via CCJP, storage time, and temperature play important roles in the inhibition of toxin formation by proteolytic C. botulinum.
In-capillary self-assembly and proteolytic cleavage of polyhistidine peptide capped quantum dots
Energy Technology Data Exchange (ETDEWEB)
Wang, Jianhao; Li, Jingyan; Li, Jinchen; Liu, Feifei [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Zhou, Xiang; Yao, Yi [Changzhou Qianhong Bio-pharma Co. Ltd, Changzhou 213164, Jiangsu (China); Wang, Cheli [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Qiu, Lin, E-mail: linqiupjj@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); Jiang, Pengju, E-mail: pengju.jiang@gmail.com [School of Pharmaceutical Engineering and Life Science, Changzhou University, Changzhou, Jiangsu, 213164 (China); State Key Laboratory of Pharmaceutical Biotechnology, Nanjing, Jiangsu (China)
2015-10-01
A new method using fluorescence coupled capillary electrophoresis (CE-FL) for monitoring self-assembly and proteolytic cleavage of hexahistidine peptide capped quantum dots (QDs) inside a capillary has been developed in this report. QDs and the ATTO 590-labeled hexahistidine peptide (H6-ATTO) were injected into a capillary, sequentially. Their self-assembly inside the capillary was driven by a metal-affinity force which yielded a new fluorescence signal due to Förster resonance energy transfer (FRET). The highly efficient separation of fluorescent complexes and the FRET process were analyzed using CE-FL. The self-assembly of QDs and biomolecules was found to effectively take place inside the capillary. The kinetics of the assembly was monitored by CE-FL, and the approach was extended to the study of proteolytic cleavage of surface conjugated peptides. Being the first in-depth analysis of in-capillary nanoparticle–biomolecule assembly, the novel approach reported here provides inspiration to the development of QD-based FRET probes for biomedical applications. - Highlights: • We examined the self-assembly QDs with H6-ATTO inside a capillary. • We prove CE-FL to be a powerful method to resolve QDs-H6-ATTO complex. • We achieve chromatographic separation of QDs-H6-ATTO complex. • We discovered a novel strategy for the online detection of thrombin. • This technique integrated “injection, mixing, reaction, separation and detection”.
Calsyntenin-1 shelters APP from proteolytic processing during anterograde axonal transport
Directory of Open Access Journals (Sweden)
Martin Steuble
2012-06-01
Endocytosis of amyloid-β precursor protein (APP is thought to represent the major source of substrate for the production of the amyloidogenic Aβ peptide by the β-secretase BACE1. The irreversible nature of proteolytic cleavage implies the existence of an efficient replenishment route for APP from its sites of synthesis to the cell surface. We recently found that APP exits the trans-Golgi network in intimate association with calsyntenin-1, a transmembrane cargo-docking protein for Kinesin-1-mediated vesicular transport. Here we characterized the function of calsyntenin-1 in neuronal APP transport using selective immunoisolation of intracellular trafficking organelles, immunocytochemistry, live-imaging, and RNAi. We found that APP is co-transported with calsyntenin-1 along axons to early endosomes in the central region of growth cones in carriers that exclude the α-secretase ADAM10. Intriguingly, calsyntenin-1/APP organelles contained BACE1, suggesting premature cleavage of APP along its anterograde path. However, we found that APP contained in calsyntenin-1/APP organelles was stable. We further analyzed vesicular trafficking of APP in cultured hippocampal neurons, in which calsyntenin-1 was reduced by RNAi. We found a markedly increased co-localization of APP and ADAM10 in axons and growth cones, along with increased proteolytic processing of APP and Aβ secretion in these neurons. This suggested that the reduced capacity for calsyntenin-1-dependent APP transport resulted in mis-sorting of APP into additional axonal carriers and, therefore, the premature encounter of unprotected APP with its ectodomain proteases. In combination, our results characterize calsyntenin-1/APP organelles as carriers for sheltered anterograde axonal transport of APP.
INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS
Bartoloni, Fernando Heering; Monteiro Leite Ciscato, Luiz Francisco; Augusto, Felipe Alberto; Baader, Wilhelm Josef
2010-01-01
INVERSE ELECTRON TRANSFER IN PEROXYOXALATE CHEMIEXCITATION USING EASILY REDUCIBLE ACTIVATORS. Chemiluminescence properties of the peroxyoxalate reaction in the presence of activators bearing electron withdrawing substituents were studied, to evaluate the possible occurrence of an inverse electron
Structural complexities in the active layers of organic electronics.
Lee, Stephanie S; Loo, Yueh-Lin
2010-01-01
The field of organic electronics has progressed rapidly in recent years. However, understanding the direct structure-function relationships between the morphology in electrically active layers and the performance of devices composed of these materials has proven difficult. The morphology of active layers in organic electronics is inherently complex, with heterogeneities existing across multiple length scales, from subnanometer to micron and millimeter range. A major challenge still facing the organic electronics community is understanding how the morphology across all of the length scales in active layers collectively determines the device performance of organic electronics. In this review we highlight experiments that have contributed to the elucidation of structure-function relationships in organic electronics and also point to areas in which knowledge of such relationships is still lacking. Such knowledge will lead to the ability to select active materials on the basis of their inherent properties for the fabrication of devices with prespecified characteristics.
DEFF Research Database (Denmark)
Behrendt, Niels; List, Karin; Andreasen, Peter A
2003-01-01
The reciprocal pro-enzyme activation system of plasmin, urokinase-type plasminogen activator (uPA) and their respective zymogens is a potent mechanism in the generation of extracellular proteolytic activity. Plasminogen activator inhibitor type 1 (PAI-1) acts as a negative regulator. This system...... is complicated by a poorly understood intrinsic reactivity of the uPA pro-enzyme (pro-uPA) before proteolytic activation, directed against both plasminogen and PAI-1. We have studied the integrated activation mechanism under the repression of PAI-1 in a purified system. A covalent reaction between pro...
Proteolytic processing of the vitellogenin precursor in the boll weevil, Anthonomus grandis.
Heilmann, L J; Trewitt, P M; Kumaran, A K
1993-01-01
The soluble proteins of the eggs of the coleopteran insect Anthonomus grandis Boheman, the cotton boll weevil, consist almost entirely of two vitellin types with M(r)s of 160,000 and 47,000. We sequenced their N-terminal ends and one internal cyanogen bromide fragment of the large vitellin and compared these sequences with the deduced amino acid sequence from the vitellogenin gene. The results suggest that both the boll weevil vitellin proteins are products of the proteolytic cleavage of a single precursor protein. The smaller 47,000 M(r) vitellin protein is derived from the N-terminal portion of the precursor adjacent to an 18 amino acid signal peptide. The cleavage site between the large and small vitellins at amino acid 362 is adjacent to a pentapeptide sequence containing two pairs of arginine residues. Comparison of the boll weevil sequences with limited known sequences from the single 180,000 M(r) honey bee protein show that the honey bee vitellin N-terminal exhibits sequence homology to the N-terminal of the 47,000 M(r) boll weevil vitellin. Treatment of the vitellins with an N-glycosidase results in a decrease in molecular weight of both proteins, from 47,000 to 39,000 and from 160,000 to 145,000, indicating that about 10-15% of the molecular weight of each vitellin consists of N-linked carbohydrate. The molecular weight of the deglycosylated large vitellin is smaller than that predicted from the gene sequence, indicating possible further proteolytic processing at the C-terminal of that protein.
Locked and proteolysis-based transcription activator-like effector (TALE) regulation.
Lonzarić, Jan; Lebar, Tina; Majerle, Andreja; Manček-Keber, Mateja; Jerala, Roman
2016-02-18
Development of orthogonal, designable and adjustable transcriptional regulators is an important goal of synthetic biology. Their activity has been typically modulated through stimulus-induced oligomerization or interaction between the DNA-binding and activation/repression domain. We exploited a feature of the designable Transcription activator-like effector (TALE) DNA-binding domain that it winds around the DNA which allows to topologically prevent it from binding by intramolecular cyclization. This new approach was investigated through noncovalent ligand-induced cyclization or through a covalent split intein cyclization strategy, where the topological inhibition of DNA binding by cyclization and its restoration by a proteolytic release of the topologic constraint was expected. We show that locked TALEs indeed have diminished DNA binding and regain full transcriptional activity by stimulation with the rapamycin ligand or site-specific proteolysis of the peptide linker, with much higher level of activation than rapamycin-induced heterodimerization. Additionally, we demonstrated reversibility, activation of genomic targets and implemented logic gates based on combinations of protein cyclization, proteolytic cleavage and ligand-induced dimerization, where the strongest fold induction was achieved by the proteolytic cleavage of a repression domain from a linear TALE. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.
Effect of purified, soluble urokinase receptor on the plasminogen-prourokinase activation system
DEFF Research Database (Denmark)
Behrendt, N; Danø, K
1996-01-01
The extracellular proteolytic pathway mediated by the urokinase plasminogen activator (uPA) is a cascade system, initiated by activation of the zymogen, pro-uPA. Pro-uPA as well as uPA binds to the cellular uPA receptor (uPAR) which has a central function in cell-dependent acceleration of the cas......The extracellular proteolytic pathway mediated by the urokinase plasminogen activator (uPA) is a cascade system, initiated by activation of the zymogen, pro-uPA. Pro-uPA as well as uPA binds to the cellular uPA receptor (uPAR) which has a central function in cell-dependent acceleration...
Top-down proteomics for the analysis of proteolytic events - Methods, applications and perspectives.
Tholey, Andreas; Becker, Alexander
2017-11-01
Mass spectrometry based proteomics is an indispensable tool for almost all research areas relevant for the understanding of proteolytic processing, ranging from the identification of substrates, products and cleavage sites up to the analysis of structural features influencing protease activity. The majority of methods for these studies are based on bottom-up proteomics performing analysis at peptide level. As this approach is characterized by a number of pitfalls, e.g. loss of molecular information, there is an ongoing effort to establish top-down proteomics, performing separation and MS analysis both at intact protein level. We briefly introduce major approaches of bottom-up proteomics used in the field of protease research and highlight the shortcomings of these methods. We then discuss the present state-of-the-art of top-down proteomics. Together with the discussion of known challenges we show the potential of this approach and present a number of successful applications of top-down proteomics in protease research. This article is part of a Special Issue entitled: Proteolysis as a Regulatory Event in Pathophysiology edited by Stefan Rose-John. Copyright © 2017 Elsevier B.V. All rights reserved.
Tsai, Shih-Jen
2017-12-22
Major depressive disorder is a common illness worldwide, but the pathogenesis of the disorder remains incompletely understood. The tissue-type plasminogen activator-plasminogen proteolytic cascade is highly expressed in the brain regions involved in mood regulation and neuroplasticity. Accumulating evidence from animal and human studies suggests that tissue-type plasminogen activator and its chief inhibitor, plasminogen activator inhibitor-1, are related to stress reaction and depression. Furthermore, the neurotrophic hypothesis of depression postulates that compromised neurotrophin brain-derived neurotrophic factor (BDNF) function is directly involved in the pathophysiology of depression. In the brain, the proteolytic cleavage of proBDNF, a BDNF precursor, to mature BDNF through plasmin represents one mechanism that can change the direction of BDNF action. We also discuss the implications of tissue-type plasminogen activator and plasminogen activator inhibitor-1 alterations as biomarkers for major depressive disorder. Using drugs that increase tissue-type plasminogen activator or decrease plasminogen activator inhibitor-1 levels may open new avenues to develop conceptually novel therapeutic strategies for depression treatment.
Activity coefficients of electrons and holes in semiconductors
International Nuclear Information System (INIS)
Orazem, M.E.; Newman, J.
1984-01-01
Dilute-solution transport equations with constant activity coefficients are commonly used to model semiconductors. These equations are consistent with a Boltzmann distribution and are invalid in regions where the species concentration is close to the respective site concentration. A more rigorous treatment of transport in a semiconductor requires activity coefficients which are functions of concentration. Expressions are presented for activity coefficients of electrons and holes in semiconductors for which conduction- and valence-band energy levels are given by the respective bandedge energy levels. These activity coefficients are functions of concentration and are thermodynamically consistent. The use of activity coefficients in macroscopic transport relationships allows a description of electron transport in a manner consistent with the Fermi-Dirac distribution
Directory of Open Access Journals (Sweden)
Gottschall Paul E
2005-08-01
Full Text Available Abstract Background Brevican is a member of the lectican family of aggregating extracellular matrix (ECM proteoglycans that bear chondroitin sulfate (CS chains. It is highly expressed in the central nervous system (CNS and is thought to stabilize synapses and inhibit neural plasticity and as such, neuritic or synaptic remodeling would be less likely to occur in regions with intact and abundant, lectican-containing, ECM complexes. Neural plasticity may occur more readily when these ECM complexes are broken down by endogenous proteases, the ADAMTSs (adisintegrin and metalloproteinase with thrombospondin motifs, that selectively cleave the lecticans. The purpose of these experiments was to determine whether the production of brevican or the ADAMTS-cleaved fragments of brevican were altered after deafferentation and reinnervation of the dentate gyrus via entorhinal cortex lesion (ECL. Results In the C57Bl6J mouse, synaptic density in the molecular layer of the dentate gyrus, as measured by synaptophysin levels in ELISA, was significantly attenuated 2 days (nearly 50% of contralateral and 7 days after lesion and returned to levels not different from the contralateral region at 30 days. Immunoreactive brevican in immunoblot was elevated 2 days after lesion, whereas there was a significant increase in the proteolytic product at 7, but not 30 days post-lesion. ADAMTS activity, estimated using the ratio of the specific ADAMTS-derived brevican fragment and intact brevican levels was increased at 7 days, but was not different from the contralateral side at 2 or 30 days after deafferentation. Conclusion These findings indicate that ADAMTS activity in the dentate outer molecular layer (OML is elevated during the initial synaptic reinnervation period (7 days after lesion. Therefore, proteolytic processing of brevican appears to be a significant extracellular event in the remodeling of the dentate after EC lesion, and may modulate the process of sprouting and
Proteolytic activities in fillets of selected underutilized Australian fish species.
Ahmed, Z; Donkor, O; Street, W A; Vasiljevic, T
2013-09-01
The hydrolytic activity of major endogenous proteases, responsible for proteolysis of myofibrillar proteins during post-mortem storage, may be an indicator of the textural quality of fish which influences consumer purchasing behaviour and thus market value of the final product. Furthermore, it may also influence the type and bioactive properties of the peptides released during post-mortem proteolysis of myofibrillar proteins. This study compared the activities of cathepsins B, B+L, D, H and calpain-like enzymes in crude muscle extracted from 16 Australian underutilized fish species. Fish species had a significant effect on the activity of these enzymes with barracouta showing the highest cathepsins B, B+L, D and H activities. Activities of cathepsins B and B+L were higher than cathepsin H for all studied species. The more commercially important rock ling and tiger flathead demonstrated higher cathepsin B+L activity, whereas gemfish and eastern school whiting showed higher activity towards cathepsin B. Underutilized fish species showing higher endogenous protease activities may be suitable for fish sauce production, whereas those with lower protease activities for surimi processing. Copyright © 2013 Elsevier Ltd. All rights reserved.
Quality control of the documentation process in electronic economic activities
Directory of Open Access Journals (Sweden)
Krutova A.S.
2017-06-01
Full Text Available It is proved that the main tool that will provide adequate information resources e economic activities of social and economic relations are documenting quality control processes as the basis of global information space. Directions problems as formation evaluation information resources in the process of documentation, namely development tools assess the efficiency of the system components – qualitative assessment; development of mathematical modeling tools – quantitative evaluation. A qualitative assessment of electronic documentation of economic activity through exercise performance, efficiency of communication; document management efficiency; effectiveness of flow control operations; relationship management effectiveness. The concept of quality control process documents electronically economic activity to components which include: the level of workflow; forms adequacy of information; consumer quality documents; quality attributes; type of income data; condition monitoring systems; organizational level process documentation; attributes of quality, performance quality consumer; type of management system; type of income data; condition monitoring systems. Grounded components of the control system electronic document subjects of economic activity. Detected components IT-audit management system economic activity: compliance audit; audit of internal control; detailed multilevel analysis; corporate risk assessment methodology. The stages and methods of processing electronic transactions economic activity during condition monitoring of electronic economic activity.
Study on regeneration of activated carbon by means of electron radiation
International Nuclear Information System (INIS)
Zhu Guanghua; Arai, H.; Hosono, M.
1991-01-01
The results of regeneration of activated carbon adsorbing sodium lauryl sulfate (SLS) by 2 MeV electron radiation, and the dependence of the regeneration rate of activated carbon on the electron current intensity, the temperature of sample and the atmosphere were reported. It is shown that regeneration of activated carbon by electron radiation is full of promise
Layat, Elodie; Cotterell, Sylviane; Vaillant, Isabelle; Yukawa, Yasushi; Tutois, Sylvie; Tourmente, Sylvette
2012-07-01
Ribosome biogenesis is critical for eukaryotic cells and requires coordinated synthesis of the protein and rRNA moieties of the ribosome, which are therefore highly regulated. 5S ribosomal RNA, an essential component of the large ribosomal subunit, is transcribed by RNA polymerase III and specifically requires transcription factor IIIA (TFIIIA). To obtain insight into the regulation of 5S rRNA transcription, we have investigated the expression of 5S rRNA and the exon-skipped (ES) and exon-including (EI) TFIIIA transcripts, two transcript isoforms that result from alternative splicing of the TFIIIA gene, and TFIIIA protein amounts with respect to requirements for 5S rRNA during development. We show that 5S rRNA quantities are regulated through distinct but complementary mechanisms operating through transcriptional and post-transcriptional control of TFIIIA transcripts as well as at the post-translational level through proteolytic cleavage of the TFIIIA protein. During the reproductive phase, high expression of the TFIIIA gene together with low proteolytic cleavage contributes to accumulation of functional, full-length TFIIIA protein, and results in 5S rRNA accumulation in the seed. In contrast, just after germination, the levels of TFIIIA-encoding transcripts are low and stable. Full-length TFIIIA protein is undetectable, and the level of 5S rRNA stored in the embryo progressively decreases. After day 4, in correlation with the reorganization of 5S rDNA chromatin to a mature state, full-length TFIIIA protein with transcriptional activity accumulates and permits de novo transcription of 5S rRNA. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.
Uchiyama, Kazuhiko; Sakiyama, Toshio; Hasebe, Takumu; Musch, Mark W.; Miyoshi, Hiroyuki; Nakagawa, Yasushi; He, Tong-Chuan; Lichtenstein, Lev; Naito, Yuji; Itoh, Yoshito; Yoshikawa, Toshikazu; Jabri, Bana; Stappenbeck, Thaddeus; Chang, Eugene B.
2016-01-01
Proliferation and spatial development of colonic epithelial cells are highly regulated along the crypt vertical axis, which, when perturbed, can result in aberrant growth and carcinogenesis. In this study, two key factors were identified that have important and counterbalancing roles regulating these processes: pericrypt myofibroblast-derived Wnt-5a and the microbial metabolite butyrate. Cultured YAMC cell proliferation and heat shock protein induction were analzyed after butryate, conditioned medium with Wnt5a activity, and FrzB containing conditioned medium. In vivo studies to modulate Hsp25 employed intra-colonic wall Hsp25 encoding lentivirus. To silence Wnt-5a in vivo, intra-colonic wall Wnt-5a silencing RNA was used. Wnt-5a, secreted by stromal myofibroblasts of the lower crypt, promotes proliferation through canonical β-catenin activation. Essential to this are two key requirements: (1) proteolytic conversion of the highly insoluble ~40 kD Wnt-5a protein to a soluble 36 mer amino acid peptide that activates epithelial β-catenin and cellular proliferation, and (2) the simultaneous inhibition of butyrate-induced Hsp25 by Wnt-5a which is necessary to arrest the proliferative process in the upper colonic crypt. The interplay and spatial gradients of these factors insures that crypt epithelial cell proliferation and development proceed in an orderly fashion, but with sufficient plasticity to adapt to physiological perturbations including inflammation. PMID:27561676
Miyamoto, Mari; Ueno, Hiroshi M; Watanabe, Masayuki; Tatsuma, Yumi; Seto, Yasuyuki; Miyamoto, Taku; Nakajima, Hadjime
2015-03-16
Airag is a traditional fermented milk of Mongolia that is usually made from raw mare's milk. Lactobacillus helveticus is one of the lactic acid bacteria most frequently isolated from airag. In this study, we investigated the genetic and physiological characteristics of L. helveticus strains isolated from airag and clarified their significance in airag by comparing them with strains from different sources. Six strains of L. helveticus were isolated from five home-made airag samples collected from different regions of Mongolia. The optimal temperature for acidification in skim milk was 30 to 35°C for all the Mongolian strains, which is lower than those for the reference strains (JCM 1554 and JCM 1120(T)) isolated from European cheeses. All of the strains had a prtH1-like gene encoding a variant type of cell envelope proteinase (CEP). The CEP amino acid sequence in Snow Brand Typeculture (SBT) 11087 isolated from airag shared 71% identity with PrtH of L. helveticus CNRZ32 (AAD50643.1) but 98% identity with PrtH of Lactobacillus kefiranofaciens ZW3 (AEG40278.1) isolated from a traditional fermented milk in Tibet. The proteolytic activities of the CEP from SBT11087 on artificial substrate (N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide) and pure casein were measured using an intact-cell degradation assay. The activity of the CEP from SBT11087 was observed to be weak and exhibited a lower optimal temperature (40°C) than those from the reference strains (45-50°C). The specificity of the SBT11087 CEP for αS1-casein was typical of the CEPs previously reported in L. helveticus, as determined through the degradation profiles obtained through gel electrophoresis and mass spectrometry analyses. In contrast, the degradation profile of β-casein revealed that the CEP of SBT11087 primarily hydrolyzes its C-terminal domain and hydrolyzed nine of the 16 cleavage sites shared among the CEPs of other L. helveticus strains. Thus, the CEP of SBT11087 is distinct from those from
Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou
2015-01-01
The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (PLactobacillus bulgaricus at the transcription level. PMID:25845365
Hou, Jun-cai; Liu, Fei; Ren, Da-xi; Han, Wei-wei; Du, Yue-ou
2015-04-01
The proteolytic system of Lactobacillus bulgaricus breaks down milk proteins into peptides and amino acids, which are essential for the growth of the bacteria. The aim of this study was to determine the expressions of seven key genes in the proteolytic system under different culturing conditions (different phases, initial pH values, temperatures, and nitrogen sources) using real-time polymerase chain reaction (RT-PCR). The transcriptions of the seven genes were reduced by 30-fold on average in the stationary phase compared with the exponential growth phase. The transcriptions of the seven genes were reduced by 62.5-, 15.0-, and 59.0-fold in the strains KLDS 08006, KLDS 08007, and KLDS 08012, respectively, indicating that the expressions of the seven genes were significantly different among strains. In addition, the expressions of the seven genes were repressed in the MRS medium containing casein peptone. The effect of peptone supply on PepX transcription was the weakest compared with the other six genes, and the impact on OppD transcription was the strongest. Moreover, the expressions of the seven genes were significantly different among different strains (Pproteolytic system genes in Lactobacillus bulgaricus at the transcription level.
Directory of Open Access Journals (Sweden)
Annaïg Mahé
2004-01-01
Full Text Available In this article, which reports the second part of a two-part study of the use of electronic journals by researchers in two French research institutions, we attempt to explain the integration of the use of electronic journals in the scientists' information habits, going beyond usage analysis. First, we describe how the development of electronic journals use follows a three-phase innovation process - research-development, first uses, and technical acculturation. Then, we attempt to find more significant explanatory factors, and emphasis is placed on the wider context of information activity. Three main information activity types are outlined - marginal, parallel, and integrated. Each of these types corresponds to a particular attitude towards scientific information and to different levels of electronic journal use.
Selection and characterization of camelid nanobodies towards urokinase-type plasminogen activator
DEFF Research Database (Denmark)
Kaczmarek, Jakub; Skottrup, Peter Durand
2015-01-01
Urokinase-type plasminogen activator (uPA) is a trypsin-like serine protease that plays a vital role in extracellular conversion of inactive plasminogen into catalytically active plasmin. Activated plasmin facilitates the release of several proteolytic enzymes, which control processes like perice...
Chen, Hui-Ling; Lünsdorf, Heinrich; Hecht, Hans-Jürgen; Tsai, Hsin
2010-08-01
The somatic angiotensin I-converting enzyme (sACE; peptidyl-dipeptidase A; EC 3.4.15.1) was isolated from pig lung and purified to homogeneity. The purified enzyme has a molecular mass of about 180 kDa. Upon proteolytic cleavage, two approximately 90 kDa fragments were obtained and identified by amino-terminal sequence analysis as the N- and C-domains of sACE. Both purified domains were shown to be catalytically active. A 2.3 nm resolution model of sACE was obtained by three-dimensional electron microscopic reconstruction of negatively stained sACE particles, based on atomic X-ray data fitting. Our model shows for the first time the relative orientation of the sACE catalytically active domains and their spatial distance. (c) 2010 Elsevier Ltd. All rights reserved.
Human NLRP3 inflammasome activation is Nox1-4 independent
van Bruggen, Robin; Köker, M. Yavuz; Jansen, Machiel; van Houdt, Michel; Roos, Dirk; Kuijpers, Taco W.; van den Berg, Timo K.
2010-01-01
The NLRP3 inflammasome can be activated by pathogen-associated molecular patterns or endogenous danger-associated molecular patterns. The activation of the NLRP3 inflammasome results in proteolytic activation and secretion of cytokines of the interleukin-1 (IL-1) family. The precise mode of
DEFF Research Database (Denmark)
Veidal, Sanne S.; Karsdal, Morten A.; Nawrocki, Arkadiusz
2011-01-01
Collagen deposition and an altered matrix metalloproteinase (MMP) expression profile are hallmarks of fibrosis. Type IV collagen is the most abundant structural basement membrane component of tissue, which increases 14-fold during fibrogenesis in the liver. Proteolytic degradation of collagens...
Oxide-cathode activation and surface temperature calculation of electron cooler
International Nuclear Information System (INIS)
Li Jie; Yang Xiaodong; Mao Lijun; Li Guohong; Yuan Youjin; Liu Zhanwen; Zhang Junhui; Yang Xiaotian; Ma Xiaoming; Yan Tailai
2011-01-01
The pollution on electron gun ceramic insulation of electron cooler restricted the operation of electron cooler at HIRFL-CSR main ring. To cool and accumulate ion beam well, the pollution was cleared and a new oxide-coated cathode was assembled. The processes of cathode replacement,vacuum chamber baking-out, and thermal decomposition of coating binders and alkaline earth metal carbonates, and cathode activation are presented. The electron gun perveance of 10.6 μA/V 1.5 was attained under the heating power of 60 W. The typical surface temperature of oxide-coated cathode that is calculated through grey-body radiation is 1 108 K which shows a comparable result to the experimental measurement 1 078 K. The perveance growth of electron gun during the electron cooler operation is also explained by partial activation of the cathode. (authors)
Directory of Open Access Journals (Sweden)
Antonio Felicioli
2013-12-01
Full Text Available Understanding the mechanism of metabolic digestion and of food assimilation in wild snake species is of fundamental importance to compare trophic niches, their adaptive significance and evolutionary patterns of diet selection for different snake species. From two adult Hierophis viridiflavus males in fasting condition, we sampled gastric fluids using fiberoptic endoscopy. We analyzed the proteolytic activities (optimal pH and temperature, response to inhibitors of snake samples by zymography techniques. The two major protease activity bands were always detected at every tested pH. Activity bands were also detectable over 37 °C; in particular both bands showed the highest activity ranging from 55 °C to 60 °C. Ranging from 65 °C to 85 °C, one band remained visible while the other band disappeared over 60 °C. The pattern of proteolytic enzymes activity of sampled gastric fluids highlighted a scenario composed of few active proteases, reflecting the feeding status (e.g., fasting of studied snakes. Detection of proteolytic activity until 85 °C, as in prokaryotic organisms, supported the hypothesis about the presence of proteases of exogenous source.
Tsai, Shih-Jen
2017-01-01
Major depressive disorder is a common illness worldwide, but the pathogenesis of the disorder remains incompletely understood. The tissue-type plasminogen activator-plasminogen proteolytic cascade is highly expressed in the brain regions involved in mood regulation and neuroplasticity. Accumulating evidence from animal and human studies suggests that tissue-type plasminogen activator and its chief inhibitor, plasminogen activator inhibitor-1, are related to stress reaction and depression. Fur...
Dulka, Brooke N.; Ford, Ellen C.; Lee, Melissa A.; Donnell, Nathaniel J.; Goode, Travis D.; Prosser, Rebecca; Cooper, Matthew A.
2016-01-01
Brain-derived neurotrophic factor (BDNF) is essential for memory processes. The present study tested whether proteolytic cleavage of proBDNF into mature BDNF (mBDNF) within the basolateral amygdala (BLA) regulates the consolidation of defeat-related memories. We found that acute social defeat increases the expression of mBDNF, but not proBDNF, in…
Dynamics of digestive proteolytic system during blood feeding of the hard tick Ixodes ricinus
Directory of Open Access Journals (Sweden)
Sojka Daniel
2010-12-01
Full Text Available Abstract Background Ticks are vectors of a wide variety of pathogens causing severe diseases in humans and domestic animals. Intestinal digestion of the host blood is an essential process of tick physiology and also a limiting factor for pathogen transmission since the tick gut represents the primary site for pathogen infection and proliferation. Using the model tick Ixodes ricinus, the European Lyme disease vector, we have previously demonstrated by genetic and biochemical analyses that host blood is degraded in the tick gut by a network of acidic peptidases of the aspartic and cysteine classes. Results This study reveals the digestive machinery of the I. ricinus during the course of blood-feeding on the host. The dynamic profiling of concentrations, activities and mRNA expressions of the major digestive enzymes demonstrates that the de novo synthesis of peptidases triggers the dramatic increase of the hemoglobinolytic activity along the feeding period. Overall hemoglobinolysis, as well as the activity of digestive peptidases are negligible at the early stage of feeding, but increase dramatically towards the end of the slow feeding period, reaching maxima in fully fed ticks. This finding contradicts the established opinion that blood digestion is reduced at the end of engorgement. Furthermore, we show that the digestive proteolysis is localized intracellularly throughout the whole duration of feeding. Conclusions Results suggest that the egressing proteolytic system in the early stage of feeding and digestion is a potential target for efficient impairment, most likely by blocking its components via antibodies present in the host blood. Therefore, digestive enzymes are promising candidates for development of novel 'anti-tick' vaccines capable of tick control and even transmission of tick-borne pathogens.
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Kocmierska-Grodzka, D [Akademia Medyczna, Bialystok (Poland). Zaklad Farmakologii
1976-03-01
Investigations were carried out of the hydroproteolytic activity of pancreas, small intestine and colon of rats after fractionated irradiation (5x150 R). Marked postirradiation enhancement of lipase activity was found in pancreas and duodenal part of intestine as well as an increase of B-glucuronidase and acid phosphatase activity in nearly all parts of the intestinal tissues. Fractionated irradiation resulted in an increase of pancreatic catheptic (proteolytic) activity, causing simultaneous decrease of proteolytic activity in intestine and colon. Preventive administation of Trichlorfon ten days before irradiation (10 mg or 30 mg/kg) evoked modification of hydroproteolytic activity in intestinal tissues of healthy and irradiated rats. 30mg/kg Trichlorfon exerted antilipolytic and anticatheptic effects in pancreas and intestinal tissues of irradiated rats.
International Nuclear Information System (INIS)
Hanecak, R.; Semler, B.L.; Anderson, C.W.; Wimmer, E.
1982-01-01
Proteolytic processing of poliovirus polypeptides was examined by the addition of antibodies directed against the viral proteins P3-7c and P2-X to a cell-free translation extract prepared from infected HeLa cells. Antisera to P3-7c specifically inhibited in vitro processing at Gln-Gly pairs. Partial amino acid sequence analysis revealed a second Tyr-Gly pair that is utilized in protein processing. Neither Tyr-Gly cleavage is affected by antibody to P3-7C. Anti-P3-7c antibodies react not only with P3-7c but also with P3-6a and P3-2, two viral polypeptides NH 2 -coterminal with P3-7c. Preimmune and anti-P2-X antibodies had no effect on the processing of poliovirus proteins in vitro. The authors conclude that the activity responsible for processing poliovirus polypeptides at Gln-Gly pairs resides in the primary structure of P3-7c and not in P2-X
Wilis Ari Setyati; Muhammad Zainuddin; Person Pesona Renta
2017-01-01
Application of bacteria in bioremediation of shrimp culture ponds is one of the methods used to clean internal pollutants. This study aimed to evaluate the pathogenicity of extracellular proteolytic enzyme produced by the probiotic bacteria as bioremediation bacteria on vannamei shrimp larvae culture. There were five probiotic bacteria, which were successfully isolated from the sediments served as substrate in mangrove area. The isolated bacteria were coded in number as 13, 19, 30, 33, and 36...
Topology Optimization of an Actively Cooled Electronics Section for Downhole Tools
DEFF Research Database (Denmark)
Soprani, Stefano; Klaas Haertel, Jan Hendrik; Lazarov, Boyan Stefanov
2015-01-01
Active cooling systems represent a possible solution to the electronics overheating that occurs in wireline downhole tools operating in high temperature oil and gas wells. A Peltier cooler was chosen to maintain the downhole electronics to a tolerable temperature, but its integration into the dow......Active cooling systems represent a possible solution to the electronics overheating that occurs in wireline downhole tools operating in high temperature oil and gas wells. A Peltier cooler was chosen to maintain the downhole electronics to a tolerable temperature, but its integration......, according to the topology optimization results and assembly constraints, and compared to the optimized cases....
K63-Linked Ubiquitination in Kinase Activation and Cancer
Energy Technology Data Exchange (ETDEWEB)
Wang, Guocan [Department of Cancer Biology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Gao, Yuan [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States); Li, Liren [Department of Genomic Medicine, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Jin, Guoxiang; Cai, Zhen [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States); Chao, Jui-I [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); Department of Biological Science and Technology, National Chiao Tung University, Hsinchu, Taiwan (China); Lin, Hui-Kuan, E-mail: hklin@mdanderson.org [Department of Molecular and Cellular Oncology, The University of Texas M. D. Anderson Cancer Center, Houston, TX (United States); The University of Texas Graduate School of Biomedical Sciences at Houston, Houston, TX (United States)
2012-01-31
Ubiquitination has been demonstrated to play a pivotal role in multiple biological functions, which include cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although the role of ubiquitination in targeting proteins for proteasome-dependent degradation have been extensively studied and well-characterized, the critical non-proteolytic functions of ubiquitination, such as protein trafficking and kinase activation, involved in cell survival and cancer development, just start to emerge, In this review, we will summarize recent progresses in elucidating the non-proteolytic function of ubiquitination signaling in protein kinase activation and its implications in human cancers. The advancement in the understanding of the novel functions of ubiquitination in signal transduction pathways downstream of growth factor receptors may provide novel paradigms for the treatment of human cancers.
K63-Linked Ubiquitination in Kinase Activation and Cancer
International Nuclear Information System (INIS)
Wang, Guocan; Gao, Yuan; Li, Liren; Jin, Guoxiang; Cai, Zhen; Chao, Jui-I; Lin, Hui-Kuan
2012-01-01
Ubiquitination has been demonstrated to play a pivotal role in multiple biological functions, which include cell growth, proliferation, apoptosis, DNA damage response, innate immune response, and neuronal degeneration. Although the role of ubiquitination in targeting proteins for proteasome-dependent degradation have been extensively studied and well-characterized, the critical non-proteolytic functions of ubiquitination, such as protein trafficking and kinase activation, involved in cell survival and cancer development, just start to emerge, In this review, we will summarize recent progresses in elucidating the non-proteolytic function of ubiquitination signaling in protein kinase activation and its implications in human cancers. The advancement in the understanding of the novel functions of ubiquitination in signal transduction pathways downstream of growth factor receptors may provide novel paradigms for the treatment of human cancers.
2011-02-16
...] Agency Information Collection Activities; Proposed Collection; Comment Request; Electronic Records; Electronic Signatures AGENCY: Food and Drug Administration, HHS. ACTION: Notice. SUMMARY: The Food and Drug... relating to FDA's electronic records and electronic signatures. DATES: Submit either electronic or written...
Lázaro, Silvia; Gamarra, David; Del Val, Margarita
2015-12-01
Major histocompatibility complex class I proteins (MHC-I) load short peptides derived from proteolytic cleavage of endogenous proteins in any cell of the body, in a process termed antigen processing and presentation. When the source proteins are altered self or encoded by a pathogen, recognition of peptide/MHC-I complexes at the plasma membrane leads to CD8(+) T-lymphocyte responses that clear infections and probably underlie tumor immune surveillance. On the other hand, presentation of self peptides may cause some types of autoimmunity. The peptides that are presented determine the specificity and efficiency of pathogen clearance or, conversely, of immunopathology. In this review we highlight the growing number of peptidases which, as a by-product of their regular activity, can generate peptide epitopes for immune surveillance. These ∼20 peptidases collectively behave as a guerrilla army partnering with the regular proteasome army in generating a variety of peptides for presentation by MHC-I and thus optimally signaling infection. Copyright © 2015 The Authors. Published by Elsevier Ltd.. All rights reserved.
Proteolytic activities in yeast after UV irradiation. Pt. 2
Energy Technology Data Exchange (ETDEWEB)
Schwencke, J.; Moustacchi, E.
1982-04-01
When the levels of three common yeast proteinases in exponentially growing cells of mutants blocked in different repair pathways are compared to that of isogenic wild-type cells, it can be seen that the level of proteinase B is enhanced in the mutants whereas the levels of leucin aminopeptidase (Leu.AP) and lysine aminopeptidase (Lys.AP) are similar in all strains. As in its corresponding wild type, the level of proteinase B activity is further enhanced after UV-irradiation in a mutant blocked in excision-repair (rad1-3). In contrast, following the same treatment the level of proteinase B remains almost constant in a mutant blocked in a general error-prone repair system (rad6-1) and in a mutant defective in a more specific mutagenic repair pathway (pso2-1). Cycloheximide, an inhibitor of protein synthesis, blocks the post-UV enhancement in proteinase B activity observed in rad1-3 indicating that, as in the wild-type cells, an inducible process is involved. The levels of Lys.AP and Leu.AP are, respectively, either unaffected or only moderately increased following UV-treatment of the repair defective mutants, as in wild-type strains.
Energy Technology Data Exchange (ETDEWEB)
Chiu, W; Hosoda, J
1978-01-01
A two-dimensionally large and thin crystal has been obtained from gp32*I, a proteolytically digested product of a DNA helix-destabilizing protein coded by gene 32 in bacteriophage T4. High-resolution electron diffraction patterns (approx. 3.7 A) are recorded from both unstained and stained protein crystals embedded in glucose. The crystal is of orthorhombic space group with a = 62.9 A and b = 47.3 A.
International Nuclear Information System (INIS)
Craft, Willie Warren; Dutch, Rebecca Ellis
2005-01-01
The Hendra virus fusion (HeV F) protein is synthesized as a precursor, F 0 , and proteolytically cleaved into the mature F 1 and F 2 heterodimer, following an HDLVDGVK 109 motif. This cleavage event is required for fusogenic activity. To determine the amino acid requirements for processing of the HeV F protein, we constructed multiple mutants. Individual and simultaneous alanine substitutions of the eight residues immediately upstream of the cleavage site did not eliminate processing. A chimeric SV5 F protein in which the furin site was substituted for the VDGVK 109 motif of the HeV F protein was not processed but was expressed on the cell surface. Another chimeric SV5 F protein containing the HDLVDGVK 109 motif of the HeV F protein underwent partial cleavage. These data indicate that the upstream region can play a role in protease recognition, but is neither absolutely required nor sufficient for efficient processing of the HeV F protein
Lytic activities in coelomic fluid of Eisenia foetida and Lumbricus terrestris.
Tucková, L; Rejnek, J; Síma, P; Ondrejová, R
1986-01-01
Coelomic fluids of the two earthworm species E.foetida (E.F.) and L.terrestris (L.T.) have not only the ability to lyse various vertebrate erythrocytes but also to digest vertebrate serum proteins. Both activities are carried by different molecules since hemolysis but not proteolysis was inhibited by simple sugars. In contrary, proteolysis was blocked by PMSF which did not influence hemolysis. Coelomic fluids of E.F. digest effectively vertebrate serum proteins (PIgG, HSA) but not the proteins of L.T. coelomic fluids. The proteolytic activity was detected in approximately 40 000 mol. wt. fraction. After digestion proteolytic fragments were analyzed by immunoelectrophoresis, SDS-PAGE and TCA precipitation. Two of the fragments reacting with PIgG antisera remained intact even after 120 h digestion.
Settachaimongkon, S.; Nout, M.J.R.; Antunes Fernandes, E.C.; Hettinga, K.A.; Vervoort, J.J.M.; Hooijdonk, van A.C.M.; Zwietering, M.H.; Smid, E.J.; Valenberg, van H.J.F.
2014-01-01
Proto-cooperation between Streptococcus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus is one of the key factors that determine the fermentation process and final quality of yoghurt. In this study, the interaction between different proteolytic strains of S. thermophilus and L.
Directory of Open Access Journals (Sweden)
Taketo Wakai
Full Text Available Transcriptional negative regulation of the proteolytic system of Lactobacillus helveticus CM4 in response to amino acids seems to be very important for the control of antihypertensive peptide production; however, it remains poorly understood. A 26-kDa protein with N-terminal cystathionine β-synthase domains (CBS domain protein, which seems to be involved in the regulatory system, was purified by using a DNA-sepharose bound 300-bp DNA fragment corresponding to the upstream regions of the six proteolytic genes that are down-regulated by amino acids. The CBS domain protein bound to a DNA fragment corresponding to the region upstream of the pepV gene in response to branched chain amino acids (BCAAs. The expression of the pepV gene in Escherichia coli grown in BCAA-enriched medium was repressed when the CBS domain protein was co-expressed. These results reveal that the CBS domain protein acts as a novel type of BCAA-responsive transcriptional regulator (BCARR in L. helveticus. From comparative analysis of the promoter regions of the six proteolysis genes, a palindromic AT-rich motif, 5'-AAAAANNCTWTTATT-3', was predicted as the consensus DNA motif for the BCARR protein binding. Footprint analysis using the pepV promotor region and gel shift analyses with the corresponding short DNA fragments strongly suggested that the BCARR protein binds adjacent to the pepV promoter region and affects the transcription level of the pepV gene in the presence of BCAAs. Homology search analysis of the C-terminal region of the BCARR protein suggested the existence of a unique βαββαβ fold structure that has been reported in a variety of ACT (aspartate kinase-chorismate mutase-tyrA domain proteins for sensing amino acids. These results also suggest that the sensing of BCAAs by the ACT domain might promote the binding of the BCARR to DNA sequences upstream of proteolysis genes, which affects the gene expression of the proteolytic system in L. helveticus.
Patient activation and use of an electronic patient portal.
Ancker, Jessica S; Osorio, Snezana N; Cheriff, Adam; Cole, Curtis L; Silver, Michael; Kaushal, Rainu
2015-01-01
Electronic patient portals give patients access to personal medical data, potentially creating opportunities to improve knowledge, self-efficacy, and engagement in healthcare. The combination of knowledge, self-efficacy, and engagement has been termed activation. Our objective was to assess the relationship between patient activation and outpatient use of a patient portal. Survey. A telephone survey was conducted with 180 patients who had been given access to a portal, 113 of whom used it and 67 of whom did not. The validated patient activation measure (PAM) was administered along with questions about demographics and behaviors. Portal users were no different from nonusers in patient activation. Portal users did have higher education level and more frequent Internet use, and were more likely to have precisely 2 prescription medications than to have more or fewer. Patients who chose to use an electronic patient portal were not more highly activated than nonusers, although they were more educated and more likely to be Internet users.
Urinary serine proteases and activation of ENaC in kidney
DEFF Research Database (Denmark)
Svenningsen, Per; Andersen, Henrik; Nielsen, Lise Hald
2015-01-01
with albuminuria compatible with impaired renal Na(+) excretion: hypertension and volume retention is secondary to proteinuria in, e.g., preeclampsia and nephrotic syndrome; plasma concentrations of renin, angiotensin II, and aldosterone are frequently suppressed in proteinuric conditions, e.g., preeclampsia......Serine proteases, both soluble and cell-attached, can activate the epithelial sodium channel (ENaC) proteolytically through release of a putative 43-mer inhibitory tract from the ectodomain of the γ-subunit. ENaC controls renal Na(+) excretion and loss-of-function mutations lead to low blood...... pressure, while gain-of-function mutations lead to impaired Na(+) excretion, hypertension, and hypokalemia. We review an emerging pathophysiological concept that aberrant glomerular filtration of plasma proteases, e.g., plasmin, prostasin, and kallikrein, contributes to proteolytic activation of ENaC, both...
Jung, Se-Hui; Kong, Deok-Hoon; Park, Seoung-Woo; Kim, Young-Myeong; Ha, Kwon-Soo
2012-08-21
Peptide arrays have emerged as a key technology for drug discovery, diagnosis, and cell biology. Despite the promise of these arrays, applications of peptide arrays to quantitative analysis of enzyme kinetics have been limited due to the difficulty in obtaining quantitative information of enzymatic reaction products. In this study, we developed a new approach for the quantitative kinetics analysis of proteases using fluorescence-conjugated peptide arrays, a surface concentration-based assay with solid-phase peptide standards using dry-off measurements, and compared it with an applied concentration-based assay. For fabrication of the peptide arrays, substrate peptides of cMMP-3, caspase-3, caspase-9, and calpain-1 were functionalized with TAMRA and cysteine, and were immobilized onto amine-functionalized arrays using a heterobifunctional linker, N-[γ-maleimidobutyloxy]succinimide ester. The proteolytic activities of the four enzymes were quantitatively analyzed by calculating changes induced by enzymatic reactions in the concentrations of peptides bound to array surfaces. In addition, this assay was successfully applied for calculating the Michaelis constant (K(m,surf)) for the four enzymes. Thus, this new assay has a strong potential for use in the quantitative evaluation of proteases, and for drug discovery through kinetics studies including the determination of K(m) and V(max).
DEFF Research Database (Denmark)
Pedersen, Katrine E; Einholm, Anja P; Christensen, Anni
2003-01-01
Negatively charged organochemical inactivators of the anti-proteolytic activity of plasminogen activator inhibitor-1 (PAI-1) convert it to inactive polymers. As investigated by native gel electrophoresis, the size of the PAI-1 polymers ranged from dimers to multimers of more than 20 units. As com...
Effect of new lines of winter wheat on microbiological activity in Luvisol
Jezierska-Tys, S.; Rachoń, L.; Rutkowska, A.; Szumiło, G.
2012-02-01
The study presented in this paper was conducted under the conditions of a field experiment. Microbiological analyses were made at various stages of winter wheat plants development ie heading, milk ripeness and full ripeness. The objective of the study was to acquire knowledge on the effect of cultivation of various lines of winter wheat on the numbers of bacteria and fungi with proteolytic capabilities, on protease and urease activity, and on the rate of the processes of ammonification and nitrification. The results of conducted study demonstrated that the number of proteolytic bacteria and fungi, as well as the activity of protease and urease, and the intensity of ammonification and nitrification processes in soil depended on both the development stage and cultivated line of winter wheat.
The soluble urokinase plasminogen activator receptor and its fragments in venous ulcers
DEFF Research Database (Denmark)
Ahmad, Anwar; Saha, Prakash; Evans, Colin
2015-01-01
OBJECTIVE: Activation of proteolytic mechanisms at the cell surface through the activity of urokinase-type plasminogen activator (uPA) bound to its receptor, uPAR, is an important process in wound healing. The soluble forms of uPAR (suPAR and its fragments I, II, and III) have nonproteolytic func...
Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.
Shivaprasad, H V; Rajesh, R; Nanda, B L; Dharmappa, K K; Vishwanath, B S
2009-05-04
To validate the scientific basis of plant latex to stop bleeding on fresh cuts. Cysteine protease(s) from Asclepias curassavica (Asclepiadaceae) plant latex was assessed for pro-coagulant and thrombin like activities. A waxy material from the latex of Asclepias curassavica latex was removed by freezing and thawing. The resulted latex enzyme fraction was assayed for proteolytic activity using denatured casein as substrate. Its coagulant activity and thrombin like activity were determined using citrated plasma and pure fibrinogen, respectively. Inhibition studies were performed using specific protease inhibitors to know the type of protease. The latex enzyme fraction exhibited strong proteolytic activity when compared to trypsin and exerted pro-coagulant action by reducing plasma clotting time from 195 to 58 s whereas trypsin reduced clotting time marginally from 195 to 155 s. The pro-coagulant activity of this enzyme fraction was exerted by selectively hydrolyzing A alpha and B beta subunits of fibrinogen to form fibrin clot when pure fibrinogen was used as substrate as assessed by fibrinogen-agarose plate method and fibrinogen polymerization assay. Trypsin failed to induce any fibrin clot under similar conditions. The electrophoretic pattern of latex enzyme fraction-induced fibrin clot was very much similar to that of thrombin-induced fibrin clot and mimic thrombin like action. The proteolytic activity including thrombin like activity of Asclepias curassavica latex enzyme fraction was completely inhibited by iodoaceticacid (IAA). Cysteine proteases from Asclepias curassavica latex exhibited strong pro-coagulant action and were found to be specific in its action (Thrombin like). This could be the basis for the use of plant latex in pharmacological applications that justify their use as folk medicine.
Nur Maulida Safitri; Endang Yuli Herawati; Jue Liang Hsu
2017-01-01
The aim of this study is to isolate the antioxidative peptide from Spirulina platensis. Peptide was obtained by proteolytic digestion, ultrafiltration, fractionation by RP-HPLC, identified by LC-MS/MS—MASCOT Distiller and measured its antioxidant activity by DPPH (2.2-Diphenyl-1-picrylhydrazyl) assay. Results showed that thermolysin was the most effective enzyme to digest this algae. The active peptide Phe-Ser-Glu-Ser-Ser-Ala-Pro-Glu-Gln-His-Tyr (m/z 1281.51) was identified and synthetized, w...
DEFF Research Database (Denmark)
Schmidt-Hansen, Birgitte; Ornås, Dorte; Grigorian, Mariam
2004-01-01
with the transcriptional modulation of genes involved in the proteolytic degradation of extracellular matrix (ECM). Treatment of SVEC 4-10 with the S100A4 protein leads to the transcriptional activation of collagenase 3 (MMP-13) mRNA followed by subsequent release of the protein from the cells. Beta-casein zymography...... demonstrates enhancement of proteolytic activity associated with MMP-13. This observation indicates that extracellular S100A4 stimulates the production of ECM degrading enzymes from endothelial cells, thereby stimulating the remodeling of ECM. This could explain the angiogenic and metastasis...
Mekkes, J. R.; Le Poole, I. C.; Das, P. K.; Bos, J. D.; Westerhof, W.
1998-01-01
Wound healing can be accelerated by removing necrotic tissue. Various methods of wound debridement have been developed, including enzymatic debridement. Recently potent proteolytic enzymes were isolated from the intestine of Euphausia superba (Antarctic krill) that might be useful for degrading
Sequencing Lys-N Proteolytic Peptides by ESI and MALDI Tandem Mass Spectrometry
Dupré, Mathieu; Cantel, Sonia; Verdié, Pascal; Martinez, Jean; Enjalbal, Christine
2011-02-01
In this study, we explored the MS/MS behavior of various synthetic peptides that possess a lysine residue at the N-terminal position. These peptides were designed to mimic peptides produced upon proteolysis by the Lys-N enzyme, a metalloendopeptidase issued from a Japanese fungus Grifola frondosa that was recently investigated in proteomic studies as an alternative to trypsin digestion, as a specific cleavage at the amide X-Lys chain is obtained that provides N-terminal lysine peptide fragments. In contrast to tryptic peptides exhibiting a lysine or arginine residue solely at the C-terminal position, and are thus devoid of such basic amino acids within the sequence, these Lys-N proteolytic peptides can contain the highly basic arginine residue anywhere within the peptide chain. The fragmentation patterns of such sequences with the ESI-QqTOF and MALDI-TOF/TOF mass spectrometers commonly used in proteomic bottom-up experiments were investigated.
Directory of Open Access Journals (Sweden)
Zhitao Wan
Full Text Available The introduction of highly active antiretroviral therapy has led to a significant reduction in the morbidity and mortality of acquired immunodeficiency syndrome patients. However, the emergence of drug resistance has resulted in the failure of treatments in large numbers of patients and thus necessitates the development of new classes of anti-HIV drugs. In this study, more than 200 plant-derived small-molecule compounds were evaluated in a cell-based HIV-1 antiviral screen, resulting in the identification of a novel HIV-1 inhibitor (fangchinoline. Fangchinoline, a bisbenzylisoquinoline alkaloid isolated from Radix Stephaniae tetrandrae, exhibited antiviral activity against HIV-1 laboratory strains NL4-3, LAI and BaL in MT-4 and PM1 cells with a 50% effective concentration ranging from 0.8 to 1.7 µM. Mechanism-of-action studies showed that fangchinoline did not exhibit measurable antiviral activity in TZM-b1 cells but did inhibit the production of infectious virions in HIV-1 cDNA transfected 293T cells, which suggests that the compound targets a late event in infection cycle. Furthermore, the antiviral effect of fangchinoline seems to be HIV-1 envelope-dependent, as the production of infectious HIV-1 particles packaged with a heterologous envelope, the vesicular stomatitis virus G glycoprotein, was unaffected by fangchinoline. Western blot analysis of HIV envelope proteins expressed in transfected 293T cells and in isolated virions showed that fangchinoline inhibited HIV-1 gp160 processing, resulting in reduced envelope glycoprotein incorporation into nascent virions. Collectively, our results demonstrate that fangchinoline inhibits HIV-1 replication by interfering with gp160 proteolytic processing. Fangchinoline may serve as a starting point for developing a new HIV-1 therapeutic approach.
Soft Active Materials for Actuation, Sensing, and Electronics
Kramer, Rebecca Krone
2012-01-01
Future generations of robots, electronics, and assistive medical devices will include systems that are soft and elastically deformable, allowing them to adapt their morphology in unstructured environments. This will require soft active materials for actuation, circuitry, and sensing of deformation and contact pressure. The emerging field of soft robotics utilizes these soft active materials to mimic the inherent compliance of natural soft-bodied systems. As the elasticity of robot components ...
Effects of proteolytic enzymes and neuraminidase on the I and i erythrocyte antigen sites
International Nuclear Information System (INIS)
Doinel, C.; Ropars, C.; Salmon, C.
1978-01-01
Homogeneous cold agglutinins, purified and labelled with 125 I, have been used in a study of the effects of neuraminidase and proteolytic enzymes on the I and i reactivities of human adult erythrocytes. Measurements were made of antigen site numbers, equilibrium constants and thermodynamic parameters. There was enhanced reactivity after enzyme treatment as well as after the release of N-acetylneuraminic acid. Steric factors were shown to be of primary importance in the accessibility of the I and i antigenic determinant. After enzyme treatment, the antigenic structures became more homogeneous in their reaction with antibodies. The heterogeneity of binding constants observed with antigenic determinants of non-treated erythrocytes is probably due to the wide range of spatial distribution of these receptors within the membrane. (author)
15-year-activity of Electron Linear Accelerator Laboratory
International Nuclear Information System (INIS)
Karolczak, S.
1999-01-01
The purchase of the Russian Electron Linear Accelerator ELU-6E by Institute of Radiation Technique of Lodz Technical University in 1978 started the activity of the ELA Laboratory. The accelerator itself and many additional scientific equipment designed and built during past 15 years have became the basic investigation tool for the ITR now. The most important measuring systems based on electron beam as irradiation source are: pulse radiolysis system with detection in IR, UV and visible region of the spectra, radiation induced conductometry, Faraday chamber and computerized data acquisition and processing system
TGF-ß Regulates Cathepsin Activation during Normal and Pathogenic Development.
Flanagan-Steet, Heather; Christian, Courtney; Lu, Po-Nien; Aarnio-Peterson, Megan; Sanman, Laura; Archer-Hartmann, Stephanie; Azadi, Parastoo; Bogyo, Matthew; Steet, Richard A
2018-03-13
Cysteine cathepsins play roles during development and disease beyond their function in lysosomal protein turnover. Here, we leverage a fluorescent activity-based probe (ABP), BMV109, to track cysteine cathepsins in normal and diseased zebrafish embryos. Using this probe in a model of mucolipidosis II, we show that loss of carbohydrate-dependent lysosomal sorting alters the activity of several cathepsin proteases. The data support a pathogenic mechanism where TGF-ß signals enhance the proteolytic processing of pro-Ctsk by modulating the expression of chondroitin 4-sulfate (C4-S). In MLII, elevated C4-S corresponds with TGF-ß-mediated increases in chst11 expression. Inhibiting chst11 impairs the proteolytic activation of Ctsk and alleviates the MLII phenotypes. These findings uncover a regulatory loop between TGF-ß signaling and Ctsk activation that is altered in the context of lysosomal disease. This work highlights the power of ABPs to identify mechanisms underlying pathogenic development in living animals. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.
Assessment of microbial activity and biomass in different soils exposed to nicosulfuron
Directory of Open Access Journals (Sweden)
Ljiljana Šantrić
2014-09-01
Full Text Available The effects of the herbicide nicosulfuron on the abundance of cellulolytic and proteolytic microorganisms, activity of β-glucosidase and protease enzymes, and microbial phosphorus biomass were examined. A laboratory bioassay was set up on two types of agricultural soils differing in physicochemical properties. The following concentrations were tested: 0.3, 0.6, 3.0 and 30.0 mg a.i./kg of soil. Samples were collected 3, 7, 14, 30 and 45 days after treatment with nicosulfuron. The results showed that nicosulfuron significantly reduced the abundance of cellulolytic microorganisms in both soils, as well as microbial biomass phosphorus in sandy loam soil. The herbicide was found to stimulate β-glucosidase and protease activity in both types of soil and microbial biomass phosphorus in loamy soil. Proteolytic microorganisms remained unaffected by nicosulfuron.
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.
Lanzetti, Manuella; da Costa, Cristiane Aguiar; Nesi, Renata Tiscoski; Barroso, Marina Valente; Martins, Vanessa; Victoni, Tatiana; Lagente, Vincent; Pires, Karla Maria Pereira; e Silva, Patrícia Machado Rodrigues; Resende, Angela Castro; Porto, Luis Cristóvão; Benjamim, Cláudia Farias; Valença, Samuel Santos
2012-12-01
Our aim was to investigate the role of oxidative stress in elastase-induced pulmonary emphysema. C57BL/6 mice were subjected to pancreatic porcine elastase (PPE) instillation (0.05 or 0.5 U per mouse, i.t.) to induce pulmonary emphysema. Lungs were collected on days 7, 14, and 21 after PPE instillation. The control group was sham injected. Also, mice treated with 1% aminoguanidine (AMG) and inducible NO synthase (iNOS) knockout mice received 0.5 U PPE (i.t.), and lungs were analyzed 21 days after. We performed bronchoalveolar lavage, biochemical analyses of oxidative stress, and lung stereology and morphometry assays. Emphysema was observed histologically at 21 days after 0.5 U PPE treatment; tissues from these mice exhibited increased alveolar linear intercept and air-space volume density in comparison with the control group. TNF-α was elevated at 7 and 14 days after 0.5 U PPE treatment, concomitant with a reduction in the IL-10 levels at the same time points. Myeloperoxidase was elevated in all groups treated with 0.5 U PPE. Oxidative stress was observed during early stages of emphysema, with increased nitrite levels and malondialdehyde and superoxide dismutase activity at 7 days after 0.5 U PPE treatment. Glutathione peroxidase activity was increased in all groups treated with 0.5 U PPE. The emphysema was attenuated when iNOS was inhibited using 1% AMG and in iNOS knockout mice. Furthermore, proteolytic stimulation by PPE enhanced the expression of nitrotyrosine and iNOS, whereas the PPE+AMG group showed low expression of iNOS and nitrotyrosine. PPE stimulus also induced endothelial (e) NOS expression, whereas AMG reduced eNOS. Our results suggest that the oxidative and nitrosative stress pathways are triggered by nitric oxide production via iNOS expression in pulmonary emphysema. Copyright © 2012 Elsevier Inc. All rights reserved.
Consolato, Francesco; Maltecca, Francesca; Tulli, Susanna; Sambri, Irene; Casari, Giorgio
2018-04-09
The proteolytic processing of dynamin-like GTPase OPA1, mediated by the activity of both YME1L1 [intermembrane (i)-AAA protease complex] and OMA1, is a crucial step in the regulation of mitochondrial dynamics. OMA1 is a zinc metallopeptidase of the inner mitochondrial membrane that undergoes pre-activating proteolytic and auto-proteolytic cleavage after mitochondrial import. Here, we identify AFG3L2 [matrix (m) - AAA complex] as the major protease mediating this event, which acts by maturing the 60 kDa pre-pro-OMA1 to the 40 kDa pro-OMA1 form by severing the N-terminal portion without recognizing a specific consensus sequence. Therefore, m - AAA and i - AAA complexes coordinately regulate OMA1 processing and turnover, and consequently control which OPA1 isoforms are present, thus adding new information on the molecular mechanisms of mitochondrial dynamics and neurodegenerative diseases affected by these phenomena.This article has an associated First Person interview with the first author of the paper. © 2018. Published by The Company of Biologists Ltd.
Golyshev, Sergey A; Berkovich, Anna K; Yakovlev, Ruslan Yu; Bystrov, Dmitry M; Ivanov, Nikita M; Balandina, Galina N; Rudenskaya, Galina N
2017-07-28
Two novel conjugates of detonation nanodiamonds (dNDs) with the proteolytic enzymes chymotrypsin and papain were synthesized. The synthesis was performed via functionalization of the dNDs' surface with acidic/alkali treatment followed by carbodiimide-mediated protein binding. Covalent binding of the enzymes was confirmed by Fourier transform infrared spectrographic analysis and high-performance liquid chromatography (HPLC) amino acid analysis. HPLC also proved the preservation of the enzymes' composition during synthesis. The same assay was used to determine the binding ratios. The ratios were 12% (mass to mass) for chymotrypsin and 7.4% for papain. The enzymatic activity of the conjugates was measured using chromogenic substrates and appeared to be approximately 40% of that of the native enzymes. The optimum pH values and stability under various conditions were determined. The sizes of resulting particles were measured using dynamic light scattering and direct electron microscopic observation. The enzyme conjugates were shown to be prone to aggregation, resulting in micrometer-sized particles. The ζ-potentials were measured and found to be positive for the conjugates. The conjugated enzymes were tested for biological activity using an in vitro model of cultured transformed human epithelial cells (HeLa cell line). It was shown that dND-conjugated enzymes effectively bind to the surface of the cells and that enzymes attack exposed proteins on the plasma membrane, including cell adhesion molecules. Incubation with conjugated enzymes results in morphological changes of the cells but does not affect cell viability, as judged by monitoring the cell division index and conducting ultrastructural studies. dNDs are internalized by the cells via endocytosis, being enclosed in forming coated vesicles by chance, and they accumulate in single membrane-bound vacuoles, presumably late endosomes/phagosomes, along with multimembranous onionlike structures. The authors propose a
Activity of flavonoids through λ sub max and electron cross-section
International Nuclear Information System (INIS)
Murthy, V.R.; Sreenivasulu, M.
1997-01-01
Flavonoids are widespread groups of natural constituents and present in the drugs in the form of mono or diglycosides. Being physiologically active, flavonoids are important in the field of pharmacological and clinical applications. Not many physical properties of flavonoids have been studied so far excepting UV and NMR studies. This paper deals with the evaluation of electron ionization cross section through λ sub max, a parameter available from UV studies. Electron ionization cross section was primarily conceived to be of use in radiation chemical data, mass spectroscopic and thermodynamic studies. But later attempts to correlate electron ionization cross-section with structural and related parameters prompted the authors to derive an explicit expression relating λ sub max and electron ionization cross-section (Q). The application of this method to correlate Q through λ sub max and interpret the results in terms of chemical activity are discussed
Study on Effects of Electron Donors on Phosphine Production from Anaerobic Activated Sludge
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Jianping Cao
2017-07-01
Full Text Available The effects of different types and concentrations of electron donors (glucose, starch, methanol and sodium acetate on the formation of phosphine from anaerobic activated sludge that has been domesticated for a prolonged period were studied in small batch experiments. The results show that types and concentrations of electron donor have significant effects on the production of phosphine from anaerobic activated sludge. Among them, glucose was the most favourable electron donor, whereas sodium acetate was the least favourable electron donor for the removal of phosphorus and the production of phosphine. Higher concentrations of electron donors were more favourable for the reduction of phosphate into phosphine, and supplying more than nine times the amount of electron donor as theoretically required for the reduction of phosphate into phosphine was favourable for the production of phosphine.
Electron beam injection during active experiments. I - Electromagnetic wave emissions
Winglee, R. M.; Kellogg, P. J.
1990-01-01
The wave emissions produced in Echo 7 experiment by active injections of electron beams were investigated to determine the properties of the electromagnetic and electrostatic fields for both the field-aligned and cross-field injection in such experiments and to evaluate the sources of free energy and relative efficiencies for the generation of the VLF and HF emissions. It is shown that, for typical beam energies in active experiments, electromagnetic effects do not substantially change the bulk properties of the beam, spacecraft charging, and plasma particle acceleration. Through simulations, beam-generated whistlers; fundamental z-mode and harmonic x-mode radiation; and electrostatic electron-cyclotron, upper-hybrid, Langmuir, and lower-hybrid waves were identified. The characteristics of the observed wave spectra were found to be sensitive to both the ratio of the electron plasma frequency to the cyclotron frequency and the angle of injection relative to the magnetic field.
Metal-Organic Frameworks as Active Materials in Electronic Sensor Devices.
Campbell, Michael G; Dincă, Mircea
2017-05-12
In the past decade, advances in electrically conductive metal-organic frameworks (MOFs) and MOF-based electronic devices have created new opportunities for the development of next-generation sensors. Here we review this rapidly-growing field, with a focus on the different types of device configurations that have allowed for the use of MOFs as active components of electronic sensor devices.
Urinary plasmin activates collecting duct ENaC current in preeclampsia
DEFF Research Database (Denmark)
Buhl, KB; Friis, Ulla Glenert; Svenningsen, Per
2012-01-01
In nephrotic syndrome, plasminogen is aberrantly filtered from plasma to the urinary space and activated along the tubular system. In vitro, plasmin increases ENaC current by proteolytic cleavage of the γ-subunit. It was hypothesized that preeclampsia is associated with plasmin-dependent ability...
Biological activity of soils strongly polluted with sulfur
Energy Technology Data Exchange (ETDEWEB)
Krol, M; Maliszewska, W; Siuta, J
1972-01-01
Studies were carried out on soils strongly polluted with sulfur and acidified (to pH 1.4). The soils were subjected to an intensive liming. In field and pot experiments, the authors determined: the total quantity of bacteria, actinomycetes, fungi, azotobacter, nitrifiers, proteolytic activity of microorganisms, activity of ammonifiers and the number of sulfur-oxidizing and sulfate-reducing bacteria. It was found that intensive liming of sulfur-affected soils restored their biological activity. 8 references, 5 figures, 1 table.
Active silicon x-ray for measuring electron temperature
International Nuclear Information System (INIS)
Snider, R.T.
1994-07-01
Silicon diodes are commonly used for x-ray measurements in the soft x-ray region between a few hundred ev and 20 keV. Recent work by Cho has shown that the charge collecting region in an underbiased silicon detector is the depletion depth plus some contribution from a region near the depleted region due to charge-diffusion. The depletion depth can be fully characterized as a function of the applied bias voltage and is roughly proportional to the squart root of the bias voltage. We propose a technique to exploit this effect to use the silicon within the detector as an actively controlled x-ray filter. With reasonable silicon manufacturing methods, a silicon diode detector can be constructed in which the sensitivity of the collected charge to the impinging photon energy spectrum can be changed dynamically in the visible to above the 20 keV range. This type of detector could be used to measure the electron temperature in, for example, a tokamak plasma by sweeping the applied bias voltage during a plasma discharge. The detector samples different parts of the energy spectrum during the bias sweep, and the data collected contains enough information to determine the electron temperature. Benefits and limitations of this technique will be discussed along with comparisons to similar methods for measuring electron temperature and other applications of an active silicon x-ray filter
Bellicha, Alice; Macé, Sandrine; Oppert, Jean-Michel
2017-09-23
The prevalence of noncommunicable diseases, including those such as type 2 diabetes, obesity, dyslipidemia, and hypertension, so-called cardiometabolic diseases, is high and is increasing worldwide. Strong evidence supports the role of physical activity in management of these diseases. There is general consensus that mHealth technology, including electronic activity monitors, can potentially increase physical activity in patients, but their use in clinical settings remains limited. Practitioners' requirements when prescribing electronic activity monitors have been poorly described. The aims of this qualitative study were (1) to explore how specialist physicians prescribe electronic activity monitors to patients presenting with cardiometabolic conditions, and (2) to better understand their motivation for and barriers to prescribing such monitors. We conducted qualitative semistructured interviews in March to May 2016 with 11 senior physicians from a public university hospital in France with expertise in management of cardiometabolic diseases (type 1 and type 2 diabetes, obesity, hypertension, and dyslipidemia). Interviews lasted 45 to 60 minutes and were audiotaped, transcribed verbatim, and analyzed using directed content analysis. We report our findings following the Consolidated Criteria for Reporting Qualitative Research (COREQ) checklist. Most physicians we interviewed had never prescribed electronic activity monitors, whereas they frequently prescribed blood glucose or blood pressure self-monitoring devices. Reasons for nonprescription included lack of interest in the data collected, lack of evidence for data accuracy, concern about work overload possibly resulting from automatic data transfer, and risk of patients becoming addicted to data. Physicians expected future marketing of easy-to-use monitors that will accurately measure physical activity duration and intensity and provide understandable motivating feedback. Features of electronic activity monitors
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Lampelj Maja
2015-12-01
Full Text Available Background. Urokinase plasminogen activator (uPA and plasminogen activator inhibitor type-1 (PAI-1 play a key role in tumour invasion and metastasis. High levels of both proteolytic enzymes are associated with poor prognosis in breast cancer patients. The purpose of this study was to evaluate the correlation between traditional prognostic factors and uPA and PAI-1 expression in primary tumour of breast cancer patients.
Oliveira, Carlos Henrique de Moura; Assaid Simão, Anderson; Marcussi, Silvana
2016-01-01
Natural compounds have been widely studied with the aim of complementing antiophidic serum therapy. The present study evaluated the inhibitory potential of ascorbic acid and a vitamin complex, composed of ascorbic acid, vitamin E, and all the B-complex vitamins, on the biological activities induced by snake venoms. The effect of vitamins was evaluated on the phospholipase, proteolytic, coagulant, and fibrinogenolytic activities induced by Bothrops moojeni (Viperidae), B. jararacussu, and B. alternatus snake venoms, and the hemagglutinating activity induced by B. jararacussu venom. The vitamin complex (1:5 and 1:10 ratios) totally inhibited the fibrinogenolytic activity and partially the phospholipase activity and proteolytic activity on azocasein induced by the evaluated venoms. Significant inhibition was observed in the coagulation of human plasma induced by venoms from B. alternatus (1:2.5 and 1:5, to vitamin complex and ascorbic acid) and B. moojeni (1:2.5 and 1:5, to vitamin complex and ascorbic acid). Ascorbic acid inhibited 100% of the proteolytic activities of B. moojeni and B. alternatus on azocasein, at 1:10 ratio, the effects of all the venoms on fibrinogen, the hemagglutinating activity of B. jararacussu venom, and also extended the plasma coagulation time induced by all venoms analyzed. The vitamins analyzed showed relevant in vitro inhibitory potential over the activities induced by Bothrops venoms, suggesting their interaction with toxins belonging to the phospholipase A2, protease, and lectin classes. The results can aid further research in clarifying the possible mechanisms of interaction between vitamins and snake enzymes.
Reporters to monitor cellular MMP12 activity
Cobos-Correa, Amanda; Mall, Marcus A.; Schultz, Carsten
2010-02-01
Macrophage elastase, also called MMP12, belongs to a family of proteolytic enzymes whose best known physiological function is the remodeling of the extracellular matrix. Under certain pathological conditions, including inflammation, chronic overexpression of MMP12 has been observed and its elevated proteolytic activity has been suggested to be the cause of pulmonary emphysema. However, it was until recently impossible to monitor the activity of MMP12 under disease conditions, mainly due to a lack of detection methods. Recent development of new reporters for monitoring MMP12 activity in living cells, such as LaRee1, provided novel insights into the pathobiology of MMP12 in pulmonary inflammation.1 In the future, these reporters might contribute to improved diagnosis and in finding better treatments for chronic inflammatory lung diseases and emphysema. Our approach for visualizing MMP12 activity is based on peptidic, membrane-targeted FRET (Foerster Resonance Energy Transfer) reporters. Here we describe a set of new reporters containing different fluorophore pairs as well as modifications in the membrane-targeting lipid moiety. We studied the influence of these modifications on reporter performance and the reporter mobility on live cell membranes by FRAP (fluorescence recovery after photobleaching). Finally, we generated several new fluorescently labeled MMP inhibitors based on the peptidic reporter structures as prototypes for future tools to inhibit and monitor MMP activity at the same time.
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Rocco Rossano
Full Text Available Honey is a sweet and healthy food produced by honeybees (Apis mellifera L. from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.
Rossano, Rocco; Larocca, Marilena; Polito, Teresa; Perna, Anna Maria; Padula, Maria Carmela; Martelli, Giuseppe; Riccio, Paolo
2012-01-01
Honey is a sweet and healthy food produced by honeybees (Apis mellifera L.) from flower nectars. Using bidimensional zymography, we have detected the, until now unrevealed, proteolytic activities present in row honey samples. The resulting zymograms were specific for each type of the four unifloral honey under study, and enzymes were identified as serine proteases by the use of specific inhibitors. Further, using bidimensional electrophoresis, we have shown that honey proteases are able to degrade the major Royal Jelly proteins and in particular MRPJ-1, the protein that promotes queen differentiation in honeybees. Our findings open new perspectives for the better understanding of honeybee development, social behaviour and role in honey production. The now discovered honey proteases may influence honey properties and quality, and bidimensional zymograms might be useful to distinguish between different honey types, establish their age and floral origin, and allow honey certification.
DEFF Research Database (Denmark)
Lindholt, Jes Sanddal; Jørgensen, B; Shi, G-P
2003-01-01
plasmin is a common activator of the known proteolytic systems involved in the aneurysmal degradation, and is reported to be associated with the expansion of abdominal aortic aneurysms (AAA). The aim of this study was to study the activating pathways of plasminogen as predictors of the progression...
International Nuclear Information System (INIS)
Burri, Dominique J.; Pasquato, Antonella; Ramos da Palma, Joel; Igonet, Sebastien; Oldstone, Michael B.A.; Kunz, Stefan
2013-01-01
Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.
Energy Technology Data Exchange (ETDEWEB)
Burri, Dominique J.; Pasquato, Antonella; Ramos da Palma, Joel [Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne CH-1011 (Switzerland); Igonet, Sebastien; Oldstone, Michael B.A. [Department of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, CA 92037 (United States); Kunz, Stefan, E-mail: Stefan.Kunz@chuv.ch [Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne CH-1011 (Switzerland)
2013-02-05
Maturation of the arenavirus GP precursor (GPC) involves proteolytic processing by cellular signal peptidase and the proprotein convertase subtilisin kexin isozyme 1 (SKI-1)/site 1 protease (S1P), yielding a tripartite complex comprised of a stable signal peptide (SSP), the receptor-binding GP1, and the fusion-active transmembrane GP2. Here we investigated the roles of SKI-1/S1P processing and SSP in the biosynthesis of the recombinant GP ectodomains of lymphocytic choriomeningitis virus (LCMV) and Lassa virus (LASV). When expressed in mammalian cells, the LCMV and LASV GP ectodomains underwent processing by SKI-1/S1P, followed by dissociation of GP1 from GP2. The GP2 ectodomain spontaneously formed trimers as revealed by chemical cross-linking. The endogenous SSP, known to be crucial for maturation and transport of full-length arenavirus GPC was dispensable for processing and secretion of the soluble GP ectodomain, suggesting a specific role of SSP in the stable prefusion conformation and transport of full-length GPC.
Tonon, Jair; Cecchini, Alessandra Lourenço; Brunnquell, Cláudia Roberta; Bernardes, Sara Santos; Cecchini, Rubens; Guarnier, Flávia Alessandra
2013-01-23
Peripheral skeletal muscle is altered in patients suffering from emphysema and chronic obstructive pulmonary disease (COPD). Oxidative stress have been demonstrated to participate on skeletal muscle loss of several states, including disuse atrophy, mechanical ventilation, and chronic diseases. No evidences have demonstrated the occurance in a severity manner. We evaluated body weight, muscle loss, oxidative stress, and chymotrypsin-like proteolytic activity in the gastrocnemius muscle of emphysemic hamsters. The experimental animals had 2 different severities of lung damage from experimental emphysema induced by 20 mg/mL (E20) and 40 mg/mL (E40) papain. The severity of emphysema increased significantly in E20 (60.52 ± 2.8, p < 0.05) and E40 (52.27 ± 4.7; crossed the alveolar intercepts) groups. As compared to the control group, there was a reduction on body (171.6 ± 15.9 g) and muscle weight (251.87 ± 24.87 mg) in the E20 group (157.5 ± 10.3 mg and 230.12 ± 23.52 mg, for body and muscle weight, respectively), which was accentuated in the E40 group (137.4 ± 7.2 g and 197.87 ± 10.49 mg, for body and muscle weight, respectively). Additionally, the thiobarbituric acid reactive substances (TBARS), tert-butyl hydroperoxide-initiated chemiluminescence (CL), carbonylated proteins, and chymotrypsin-like proteolytic activity were elevated in the E40 group as compared to the E20 group (p < 0.05 for all comparisons). The severity of emphysema significantly correlated with the progressive increase in CL (r = -0.95), TBARS (r = -0.98), carbonyl proteins (r = -0.99), and chymotrypsin-like proteolytic activity (r = -0.90). Furthermore, augmentation of proteolytic activity correlated significantly with CL (r = 0.97), TBARS (r = 0.96), and carbonyl proteins (r = 0.91). Taken together, the results of the present study suggest that muscle atrophy observed in this model of emphysema is mediated by increased muscle chymotrypsin-like activity, with possible involvement of
Extracellular Protease Activity of Enteropathogenic Escherechia coli on Mucin Substrate
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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.
Artemisinin inhibits chloroplast electron transport activity: mode of action.
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Adyasha Bharati
Full Text Available Artemisinin, a secondary metabolite produced in Artemisia plant species, besides having antimalarial properties is also phytotoxic. Although, the phytotoxic activity of the compound has been long recognized, no information is available on the mechanism of action of the compound on photosynthetic activity of the plant. In this report, we have evaluated the effect of artemisinin on photoelectron transport activity of chloroplast thylakoid membrane. The inhibitory effect of the compound, under in vitro condition, was pronounced in loosely and fully coupled thylakoids; being strong in the former. The extent of inhibition was drastically reduced in the presence of uncouplers like ammonium chloride or gramicidin; a characteristic feature described for energy transfer inhibitors. The compound, on the other hand, when applied to plants (in vivo, behaved as a potent inhibitor of photosynthetic electron transport. The major site of its action was identified to be the Q(B; the secondary quinone moiety of photosystemII complex. Analysis of photoreduction kinetics of para-benzoquinone and duroquinone suggest that the inhibition leads to formation of low pool of plastoquinol, which becomes limiting for electron flow through photosystemI. Further it was ascertained that the in vivo inhibitory effect appeared as a consequence of the formation of an unidentified artemisinin-metabolite rather than by the interaction of the compound per se. The putative metabolite of artemisinin is highly reactive in instituting the inhibition of photosynthetic electron flow eventually reducing the plant growth.
LENUS (Irish Health Repository)
Boehm, Manja
2012-04-25
AbstractBackgroundCampylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear.ResultsIn the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni’s HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori’s HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria.ConclusionThese results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.
Boehm, Manja; Hoy, Benjamin; Rohde, Manfred; Tegtmeyer, Nicole; Bæk, Kristoffer T; Oyarzabal, Omar A; Brøndsted, Lone; Wessler, Silja; Backert, Steffen
2012-04-25
Campylobacter jejuni is one of the most important bacterial pathogens causing food-borne illness worldwide. Crossing the intestinal epithelial barrier and host cell entry by C. jejuni is considered the primary reason of damage to the intestinal tissue, but the molecular mechanisms as well as major bacterial and host cell factors involved in this process are still widely unclear. In the present study, we characterized the serine protease HtrA (high-temperature requirement A) of C. jejuni as a secreted virulence factor with important proteolytic functions. Infection studies and in vitro cleavage assays showed that C. jejuni's HtrA triggers shedding of the extracellular E-cadherin NTF domain (90 kDa) of non-polarised INT-407 and polarized MKN-28 epithelial cells, but fibronectin was not cleaved as seen for H. pylori's HtrA. Deletion of the htrA gene in C. jejuni or expression of a protease-deficient S197A point mutant did not lead to loss of flagella or reduced bacterial motility, but led to severe defects in E-cadherin cleavage and transmigration of the bacteria across polarized MKN-28 cell layers. Unlike other highly invasive pathogens, transmigration across polarized cells by wild-type C. jejuni is highly efficient and is achieved within a few minutes of infection. Interestingly, E-cadherin cleavage by C. jejuni occurs in a limited fashion and transmigration required the intact flagella as well as HtrA protease activity, but does not reduce transepithelial electrical resistance (TER) as seen with Salmonella, Shigella, Listeria or Neisseria. These results suggest that HtrA-mediated E-cadherin cleavage is involved in rapid crossing of the epithelial barrier by C. jejuni via a very specific mechanism using the paracellular route to reach basolateral surfaces, but does not cleave the fibronectin receptor which is necessary for cell entry.
Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.
Shrestha, Madan K; Peri, Irena; Smirnoff, Patricia; Birk, Yehudith; Golan-Goldhirsh, Avi
2002-09-25
The jojoba, Simmondsia chinensis, is a characteristic desert plant native to the Sonoran desert. The jojoba meal after oil extraction is rich in protein. The major jojoba proteins were albumins (79%) and globulins (21%), which have similar amino acid compositions and also showed a labile thrombin-inhibitory activity. SDS-PAGE showed two major proteins at 50 kDa and 25 kDa both in the albumins and in the globulins. The 25 kDa protein has trypsin- and chymotrypsin-inhibitory activities. In vitro digestibility of the globulins and albumins resembled that of casein and soybean protein concentrates and was increased after heat treatment. The increased digestibility achieved by boiling may be attributed to inactivation of the protease inhibitors and denaturation of proteins.
International Nuclear Information System (INIS)
Schaehs, Philipp; Weidinger, Petra; Probst, Olivia C.; Svoboda, Barbara; Stadlmann, Johannes; Beug, Hartmut; Waerner, Thomas; Mach, Lukas
2008-01-01
Cellular repressor of E1A-stimulated genes (CREG) has been reported to be a secretory glycoprotein implicated in cellular growth and differentiation. We now show that CREG is predominantly localized within intracellular compartments. Intracellular CREG was found to lack an N-terminal peptide present in the secreted form of the protein. In contrast to normal cells, CREG is largely secreted by fibroblasts missing both mannose 6-phosphate receptors. This is not observed in cells lacking only one of them. Mass spectrometric analysis of recombinant CREG revealed that the protein contains phosphorylated oligosaccharides at either of its two N-glycosylation sites. Cellular CREG was found to cosediment with lysosomal markers upon subcellular fractionation by density-gradient centrifugation. In fibroblasts expressing a CREG-GFP fusion construct, the heterologous protein was detected in compartments containing lysosomal proteins. Immunolocalization of endogenous CREG confirmed that intracellular CREG is localized in lysosomes. Proteolytic processing of intracellular CREG involves the action of lysosomal cysteine proteinases. These results establish that CREG is a lysosomal protein that undergoes proteolytic maturation in the course of its biosynthesis, carries the mannose 6-phosphate recognition marker and depends on the interaction with mannose 6-phosphate receptors for efficient delivery to lysosomes
Directory of Open Access Journals (Sweden)
M.E. Pereira
2005-11-01
Full Text Available The present study describes the main characteristics of the proteolytic activities of the velvetbean caterpillar, Anticarsia gemmatalis Hübner, and their sensitivity to proteinase inhibitors and activators. Midguts of last instar larvae reared on an artificial diet were homogenized in 0.15 M NaCl and centrifuged at 14,000 g for 10 min at 4ºC and the supernatants were used in enzymatic assays at 30ºC, pH 10.0. Basal total proteolytic activity (azocasein hydrolysis was 1.14 ± 0.15 absorbance variation min-1 mg protein-1, at 420 nm; basal trypsin-like activity (N-benzoyl-L-arginine-p-nitroanilide, BApNA, hydrolysis was 0.217 ± 0.02 mmol p-nitroaniline min-1 mg protein-1. The maximum proteolytic activities were observed at pH 10.5 using azocasein and at pH 10.0 using BApNA, this pH being identical to the midgut pH of 10.0. The maximum trypsin-like activity occurred at 50ºC, a temperature that reduces enzyme stability to 80 and 60% of the original, when pre-incubated for 5 and 30 min, respectively. Phenylmethylsulfonyl fluoride inhibited the proteolytic activities with an IC50 of 0.39 mM for azocasein hydrolysis and of 1.35 mM for BApNA hydrolysis. Benzamidine inhibited the hydrolysis with an IC50 of 0.69 and 0.076 mM for azocasein and BApNA, respectively. The absence of cysteine-proteinases is indicated by the fact that 2-mercaptoethanol and L-cysteine did not increase the rate of azocasein hydrolysis. These results demonstrate the presence of serine-proteinases and the predominance of trypsin-like activity in the midgut of Lepidoptera insects, now also detected in A. gemmatalis, and suggest this enzyme as a major target for pest control based on disruption of protein metabolism using proteinase inhibitors.
International Nuclear Information System (INIS)
Belov, A.A.; Ryl'tsev, V.V.; Ignatyuk, T.E.; Filatov, V.N.
1994-01-01
It is found the complex effect of the bound enzyme concentration on the proteolytic activity of trypsin immobilized to dialdehydecellulose (preriodate oxidation) after γ-irradiation and in process of storage. It is shown the occurance of three stages of immobilized enzyme inactivation in process of immobilization and storage. The velocity of inactivation did not depend on bound trypsin concentration. The ratio of proteolytic activity of samples before and after γ-irradiation was increased with the increase of immobilized to carrier enzyme concentration and was not change (in range of experiment error) in process of storage. The results were compared with that of cryctlline trypsin
International Nuclear Information System (INIS)
Afeseh Ngwa, Hilary; Kanthasamy, Arthi; Gu, Yan; Fang, Ning; Anantharam, Vellareddy; Kanthasamy, Anumantha G.
2011-01-01
The production of man-made nanoparticles for various modern applications has increased exponentially in recent years, but the potential health effects of most nanoparticles are not well characterized. Unfortunately, in vitro nanoparticle toxicity studies are extremely limited by yet unresolved problems relating to dosimetry. In the present study, we systematically characterized manganese (Mn) nanoparticle sizes and examined the nanoparticle-induced oxidative signaling in dopaminergic neuronal cells. Differential interference contrast (DIC) microscopy and transmission electron microscopy (TEM) studies revealed that Mn nanoparticles range in size from single nanoparticles (∼ 25 nM) to larger agglomerates when in treatment media. Manganese nanoparticles were effectively internalized in N27 dopaminergic neuronal cells, and they induced a time-dependent upregulation of the transporter protein transferrin. Exposure to 25–400 μg/mL Mn nanoparticles induced cell death in a time- and dose-dependent manner. Mn nanoparticles also significantly increased ROS, accompanied by a caspase-mediated proteolytic cleavage of proapoptotic protein kinase Cδ (PKCδ), as well as activation loop phosphorylation. Blocking Mn nanoparticle-induced ROS failed to protect against the neurotoxic effects, suggesting the involvement of other pathways. Further mechanistic studies revealed changes in Beclin 1 and LC3, indicating that Mn nanoparticles induce autophagy. Primary mesencephalic neuron exposure to Mn nanoparticles induced loss of TH positive dopaminergic neurons and neuronal processes. Collectively, our results suggest that Mn nanoparticles effectively enter dopaminergic neuronal cells and exert neurotoxic effects by activating an apoptotic signaling pathway and autophagy, emphasizing the need for assessing possible health risks associated with an increased use of Mn nanoparticles in modern applications. -- Highlights: ► Mn nanoparticles activate mitochondrial cell death signaling
Visualization of proteolytic activity associated with the apoptotic response in cancer cells
Tice, Brian George
Caspases execute programmed cell death, where low levels of caspase activity are linked to cancer. Chemotherapies utilize induction of apoptosis as a key mechanism for cancer treatment, where caspase-3 is a major player involved in dismantling these aberrant cells. The ability to sensitively measure the initial caspase-3 cleavage events during apoptosis is important for understanding the initiation of this complex cellular process, however, current ensemble methods are not sensitive enough to measure single cleavage events in cells. By utilizing the optical properties of plasmon coupling, peptide-linked gold nanoparticles were developed to enable single molecule imaging of caspase-3 activity in two different cancer systems. Au crown nanoparticles were assembled in a multimeric fashion to overcome the high and heterogeneous background scattering of live cells. In a colon cancer (SW620) cell line challenged with tumor necrosis factor-alpha (TNF-alpha), single molecule trajectories show early stage caspase-3 activation within minutes, which was not detectable by ensemble assays until 23 hours. Variability in caspase-3 activation among the population of cells was identified and likely a result of each cell's specific resistance to death receptor-induced apoptosis. Following these studies, improvements by way of sensitivity and selectivity were tailored into an improved nanosensor construct. Au nanoshell dimers were prepared as a comparably bright construct with 1) reduced heterogeneity compared to the synthesis of the crown nanoparticles and 2) a peptide sequence highly selective for caspase-3. Chronic myeloid leukemia (CML) K562 cells were assessed for their early apoptotic response upon treatment with dasatinib, a clinically approved tyrosine kinase inhibitor that specifically targets BCR-ABL. It has been demonstrated that inhibition of BCR-ABL by dasatinib commits K562 cells to apoptosis. Single molecule experiments with Au nanoshell dimers show caspase-3 activation
Eijsbouts, Sonja; Li, Xuang; Juan-Alcaniz, Jana; van den Oetelaar, Leon C A; Bergwerff, Jaap; Loos, Joachim; Carlsson, Anna; Vogt, E.T.C.
2017-01-01
Conventional two-dimensional (2D) transmission electron microscopy of sulfidic hydroprocessing catalysts can be deceiving and give the impression that parts of the support are overloaded with active phase. High-angle annular dark field scanning transmission electron microscopy tomography reveals
International Nuclear Information System (INIS)
Smith, A.C.; Harmon, J.M.
1987-01-01
The structural organization of the steroid-binding protein of the IM-9 cell glucocorticoid receptor was investigated by using one- and two-dimensional gel electrophoresis of proteolytic receptor fragments. One-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of receptor fragments isolated after trypsin digestion of immunopurified [ 3 H]dexamethasone 21-mesylate ([ 3 H]DM-) labeled receptor revealed the presence of a stable 26.5-kilodalton (kDa) steroid-containing non-DNA-binding fragment, derived from a larger, less stable, 29-kDa fragment. The 26.5-kDa tryptic fragment appeared to be completely contained within a 41-kDa, steroid-containing, DNA-binding species isolated after chymotrypsin digestion of the intact protein. Two-dimensional electrophoretic analysis of the [ 3 H]DM-labeled tryptic fragments resolved two 26.5-kDa and two 29-kDa components. This was the same number of isoforms seen in the intact protein, indicating that the charge heterogeneity of the steroid-binding protein is the result of modification within the steroid-containing, non-DNA-binding, 26.5-kDa tryptic fragment. Two-dimensional analysis of the 41-kDa [ 3 H]DM-labeled chymotryptic species revealed a pattern of isoforms more complex than that seen either in the intact protein or in the steroid-containing tryptic fragments. These results suggest that the 41-kDa [ 3 H]DM-labeled species resolved by one-dimensional SDS-PAGE after chymotrypsin digestion may be composed of several distinct proteolytic fragments
Proteolytic regulation of metabolic enzymes by E3 ubiquitin ligase complexes: lessons from yeast.
Nakatsukasa, Kunio; Okumura, Fumihiko; Kamura, Takumi
2015-01-01
Eukaryotic organisms use diverse mechanisms to control metabolic rates in response to changes in the internal and/or external environment. Fine metabolic control is a highly responsive, energy-saving process that is mediated by allosteric inhibition/activation and/or reversible modification of preexisting metabolic enzymes. In contrast, coarse metabolic control is a relatively long-term and expensive process that involves modulating the level of metabolic enzymes. Coarse metabolic control can be achieved through the degradation of metabolic enzymes by the ubiquitin-proteasome system (UPS), in which substrates are specifically ubiquitinated by an E3 ubiquitin ligase and targeted for proteasomal degradation. Here, we review select multi-protein E3 ligase complexes that directly regulate metabolic enzymes in Saccharomyces cerevisiae. The first part of the review focuses on the endoplasmic reticulum (ER) membrane-associated Hrd1 and Doa10 E3 ligase complexes. In addition to their primary roles in the ER-associated degradation pathway that eliminates misfolded proteins, recent quantitative proteomic analyses identified native substrates of Hrd1 and Doa10 in the sterol synthesis pathway. The second part focuses on the SCF (Skp1-Cul1-F-box protein) complex, an abundant prototypical multi-protein E3 ligase complex. While the best-known roles of the SCF complex are in the regulation of the cell cycle and transcription, accumulating evidence indicates that the SCF complex also modulates carbon metabolism pathways. The increasing number of metabolic enzymes whose stability is directly regulated by the UPS underscores the importance of the proteolytic regulation of metabolic processes for the acclimation of cells to environmental changes.
International Nuclear Information System (INIS)
Tehrani, H. Sepasi; Moosavi-Movahedi, A.A.; Ghourchian, H.
2013-01-01
Highlights: • Proline increases ET in Bovine Liver Catalase (BLC) whereas histidine decreases it. • Proline also increased the biological activity, whereas histidine decreased it. • Electron transferring and biological activity for BLC are directly correlated. • Proline causes favorable ET for BLC shown by positive E 1/2 (E°′) and negative ΔG. • Histidine makes ET unfavorable for BLC, manifested by E 1/2 (E°′) 0. -- Abstract: Catalase is a crucial antioxidant enzyme that protects life against detrimental effects of H 2 O 2 by disproportionating it into water and molecular oxygen. Effect of proline as a compatible and histidine as a non compatible osmolyte on the electron transferring and midpoint potential of catalase has been investigated. Proline increases the midpoint potential (ΔE m > 0), therefore causing the ΔG ET to be less positive and making the electron transfer reaction more facile whereas histidine decreases the E m (ΔE m ET , thereby rendering the electron transfer reaction less efficient. These results indicate the inhibitory effect of histidine evident by a −37% decrease in the cathodic peak current compared to 16% increase in the case of proline indicative of activation. The insight paves the tedious way towards our ultimate goal of elucidating a correlation between biological activity and electron transferring
Cecchini, Stefano; Caputo, Anna R
2009-01-01
Several studies have shown an immunomodulatory effect of orally administered bovine lactoferrin (LF) in fish, but the process of digestion was not characterized. In the present study, we investigated the fate of bovine LF after oral and anal administration, and studied the appearance of intact LF in the bloodstream and its proteolytic attack during the gastric transit in rainbow trout (Oncorhynchus mykiss) held at 9 degrees C and 18 degrees C. Data obtained showed the presence of intact bovine LF in the bloodstream only after anal administration in fish held at 18 degrees C and the presence of several peptides derived from bovine LF in the gastric content. Immunoblotting analysis showed that only a part of bovine LF-derived peptides reacted with the applied anti-bovine LF antibody. The concentration of intact bovine LF, after 30 min of administration, in the gastric content of fish reared at 18 degrees C, being extremely low, if any, led us to suspect that the immunoregulatory effect of dietary bovine LF shown in fish by several authors is not due to the intact form but to bioactive fragments, originated by the proteolytic attack during the gastric transit, as demonstrated in higher vertebrates.
Constitutive NADPH-dependent electron transferase activity of the Nox4 dehydrogenase domain.
Nisimoto, Yukio; Jackson, Heather M; Ogawa, Hisamitsu; Kawahara, Tsukasa; Lambeth, J David
2010-03-23
NADPH oxidase 4 (Nox4) is constitutively active, while Nox2 requires the cytosolic regulatory subunits p47(phox) and p67(phox) and activated Rac with activation by phorbol 12-myristate 13-acetate (PMA). This study was undertaken to identify the domain on Nox4 that confers constitutive activity. Lysates from Nox4-expressing cells exhibited constitutive NADPH- but not NADH-dependent hydrogen peroxide production with a K(m) for NADPH of 55 +/- 10 microM. The concentration of Nox4 in cell lysates was estimated using Western blotting and allowed calculation of a turnover of approximately 200 mol of H(2)O(2) min(-1) (mol of Nox4)(-1). A chimeric protein (Nox2/4) consisting of the Nox2 transmembrane (TM) domain and the Nox4 dehydrogenase (DH) domain showed H(2)O(2) production in the absence of cytosolic regulatory subunits. In contrast, chimera Nox4/2, consisting of the Nox4 TM and Nox2 DH domains, exhibited PMA-dependent activation that required coexpression of regulatory subunits. Nox DH domains from several Nox isoforms were purified and evaluated for their electron transferase activities. Nox1 DH, Nox2 DH, and Nox5 DH domains exhibited barely detectable activities toward artificial electron acceptors, while the Nox4 DH domain exhibited significant rates of reduction of cytochrome c (160 min(-1), largely superoxide dismutase-independent), ferricyanide (470 min(-1)), and other electron acceptors (artificial dyes and cytochrome b(5)). Rates were similar to those observed for H(2)O(2) production by the Nox4 holoenzyme in cell lysates. The activity required added FAD and was seen with NADPH but not NADH. These results indicate that the Nox4 DH domain exists in an intrinsically activated state and that electron transfer from NADPH to FAD is likely to be rate-limiting in the NADPH-dependent reduction of oxygen by holo-Nox4.
Protecting patients’ electronic health records using enhanced active bundles
Salih, R.M.; Lilien, L.T.; Ben Othmane, L.; Arriaga, R.; Matic, A.
2012-01-01
We propose a solution that provides protection for patients' electronic health/medical records disseminated among different authorized healthcare information systems. The solution is known as Active Bundles using a Trusted Third Party (ABTTP). It is based on the use of trusted third parties, and the
Physicochemical properties of elastase isolated from clinical Pseudomonas Aeruginosa
International Nuclear Information System (INIS)
Elbazza, Z.E.; Moroz, A.F.
1989-01-01
Purified elastase was obtained from clinical Pseudomonas Aeruginosa (P.A.-283). The enzyme showed not only elasto lytic activity, but also a broad proteolytic activity against various proteins. The activity of the enzyme on collagen and gelatin was also observed. The optimum pH for elastase was 7.8 to 8.0 for both the proteolytic and elasto lytic activities. The elastase was stable in a pH range from 6.6 to 9.0. Optimum temperature for proteolytic and elasto lytic activities was 40 and inhibition of elastase occurs at 80 . The D 1 0 value of the P.A-283 was found to be 0.11 kGy. Increasing the dose level value of gamma-irradiation decrease the proteolytic activity in the culture filtrate reaching only 16% at the dose level 0.5 kGy. Chelating agents and some metal ions inhibited both proteolytic and elasto lytic activities. Selective inhibition of elasto lytic activity was observed in high concentrations of sodium and ammonium salts without concurrent decrease in the proteolytic activity of the enzyme.4 fig., 3 tab
Czech Academy of Sciences Publication Activity Database
Ardan, Taras; Kocurová, Gabriela; Motlík, Jan
2015-01-01
Roč. 78, Suppl 2 (2015), s. 12-12 ISSN 1210-7859. [Conference on Animal Models for neurodegenerative Diseases /3./. 08.11.2015-10.11.2015, Liblice] R&D Projects: GA MŠk ED2.1.00/03.0124; GA MŠk(CZ) 7F14308 Institutional support: RVO:67985904 Keywords : Huntington ´s disease * transgenic porcine model * proteolytic enzymes Subject RIV: EB - Genetics ; Molecular Biology
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
Memar, Bahareh; Jamili, Shahla; Shahbazzadeh, Delavar; Bagheri, Kamran Pooshang
2016-04-01
Pterois russelli is a venomous fish belonging to scorpionidae family. Regarding to high significance value for tracing potential therapeutic molecules and special agents from venomous marine creatures, the present study was aimed to characterization of the Persian Gulf lionfish venom. Proteolytic, phospholipase, hemolytic, coagulation, edematogenic and dermonecrotic activities were determined for extracted venom. The LD50 of P. russelli venom was determined by intravenous injection in white Balb/c mice. Phospholipase A2 activity was recorded at 20 μg of total venom. Coagulation activity on human plasma was shown by Prothrombin Time (PT) and activated Partial Thromboplastin Time (APTT) assays and coagulation visualized after 7 and 14 s respectively for 60 μg of crude venom. LD50 was calculated as 10.5 mg/kg. SDS-PAGE revealed the presence of major and minor protein bands between 6 and 205 kDa. Different amounts of crude venom ranged from 1.87 to 30 μg showed proteolytic activity on casein. The highest edematic activity was detected at 20 μg. Our findings showed that the edematic activity was dose dependent and persisted for 48 h after injection. The crude venom did not induce dermonecrotic activity on rabbit skin and showed no hemolytic activity on human, mouse and rabbit erythrocytes. This is the first report for phospholipase A2 and coagulation activity in venomous fish and venomous marine animals respectively. Proteolytic activity of P. russelli venom is in accordance with the other genara of scorpionidae family. According to venom activity on intrinsic and extrinsic coagulation pathways, lionfish venom would be contained an interesting pharmaceutical agent. This study is pending to further characterization of phospholipase A2, coagulation, and protease activities and also in vivo activity on animal model of surface and internal bleeding. Copyright © 2016 Elsevier Ltd. All rights reserved.
Effect of electron beam-irradiation to b-glucan on its immunomodulating and antitumor activity
Energy Technology Data Exchange (ETDEWEB)
Jung, Yeon Hwan; Lee, Jung Lim; Yoo, Yung Choon [Konyand Univ., Daejeon (Korea, Republic of); Kim, Jae Hoon; Lee, Ju Woon [Korea Atomic Energy Research Institute, Daejeon (Korea, Republic of)
2010-07-01
In this study, in order investigated the effect of electron beam irradiation to b-glucan on its biological activities, we compared immunomodulating and antitumor activity between non-irradiated and electron beam-irradiated b-glucan. EB-glucan was irradiated by electron beam with 10, 30 and 50 kGy. Treatment with EB-glucan resulted in a slight increase of the proliferation of ConA-stimulated splenocytes, and the strongest activity was seen in 50 kGy-treated EB-glucan. EB-glucan teated with 50 kGy also showed increased secretion of cytokines such as IL-2 IFN-{gamma} and IL-6 from ConA-stimulated splenocytes. The activity of EB-glucan to enhance the proliferation of splenocytes and cytokine secretion from ConA-stimulated splenocytes was higher than that of NI-glucan. Furthermore, EB-glucan treated with 50 kGy showed higher activity to activate RAW 264.7 macrophages, comparing with that of NI-glucan. In experiments of antitumor activity, EB-glucan treated with 50 kGy prior to tumor inoculation inhibited an experimental lung metastasis produced by B16-BL6 melanoma cells in mice. But NI-glucan did show no effect. In addition, EB-glucan treated with 50 kGy induced a decrease a decrease of tumor growth in tumor-bearing mice. Collectivelt, these results indicates that electron beam irradiation {beta}-glucan leads its biological functions to enhance immunomodulating and antitumor activity.
Effect of electron beam-irradiation to b-glucan on its immunomodulating and antitumor activity
International Nuclear Information System (INIS)
Jung, Yeon Hwan; Lee, Jung Lim; Yoo, Yung Choon; Kim, Jae Hoon; Lee, Ju Woon
2010-01-01
In this study, in order investigated the effect of electron beam irradiation to b-glucan on its biological activities, we compared immunomodulating and antitumor activity between non-irradiated and electron beam-irradiated b-glucan. EB-glucan was irradiated by electron beam with 10, 30 and 50 kGy. Treatment with EB-glucan resulted in a slight increase of the proliferation of ConA-stimulated splenocytes, and the strongest activity was seen in 50 kGy-treated EB-glucan. EB-glucan teated with 50 kGy also showed increased secretion of cytokines such as IL-2 IFN-γ and IL-6 from ConA-stimulated splenocytes. The activity of EB-glucan to enhance the proliferation of splenocytes and cytokine secretion from ConA-stimulated splenocytes was higher than that of NI-glucan. Furthermore, EB-glucan treated with 50 kGy showed higher activity to activate RAW 264.7 macrophages, comparing with that of NI-glucan. In experiments of antitumor activity, EB-glucan treated with 50 kGy prior to tumor inoculation inhibited an experimental lung metastasis produced by B16-BL6 melanoma cells in mice. But NI-glucan did show no effect. In addition, EB-glucan treated with 50 kGy induced a decrease a decrease of tumor growth in tumor-bearing mice. Collectivelt, these results indicates that electron beam irradiation β-glucan leads its biological functions to enhance immunomodulating and antitumor activity
Abbott, Rebecca A; Smith, Anne J; Howie, Erin K; Pollock, Clare; Straker, Leon
2014-08-01
Active-input videogames could provide a useful conduit for increasing physical activity by improving a child's self-confidence, physical activity enjoyment, and reducing anxiety. Therefore this study evaluated the impact of (a) the removal of home access to traditional electronic games or (b) their replacement with active-input videogames, on child self-perception, enjoyment of physical activity, and electronic game use anxiety. This was a crossover, randomized controlled trial, conducted over a 6-month period in participants' family homes in metropolitan Perth, Australia, from 2007 to 2010. Children 10-12 years old were recruited through school and community media. Of 210 children who were eligible, 74 met inclusion criteria, and 8 withdrew, leaving 66 children (33 girls) for analysis. A counterbalanced randomized order of three conditions sustained for 8 weeks each: No home access to electronic games, home access to traditional electronic games, and home access to active-input electronic games. Perception of self-esteem (Harter's Self Perception Profile for Children), enjoyment of physical activity (Physical Activity Enjoyment Scale questionnaire), and anxiety toward electronic game use (modified Loyd and Gressard Computer Anxiety Subscale) were assessed. Compared with home access to traditional electronic games, neither removal of all electronic games nor replacement with active-input games resulted in any significant change to child self-esteem, enjoyment of physical activity, or anxiety related to electronic games. Although active-input videogames have been shown to be enjoyable in the short term, their ability to impact on psychological outcomes is yet to be established.
Polarized electrons and the origin of optical activity
International Nuclear Information System (INIS)
Bonner, W.A.; Dort, M.A. Van; Yearian, M.R.; Zeman, H.D.; Li, G.C.; Stanford Univ., Calif.
1976-01-01
The history of experiments bearing on the origin of optical acitivity in nature by parity non-conservation during the β-decay of radioactive isotopes is briefly reviewed. Following this, we present a more detailed description of our recent published and unpublished data and calculations regarding the generation of optical activity in DL-leucine by means of artificially produced longitudinally polarized electrons from a linear accelerator
International Nuclear Information System (INIS)
Pas, H.H.; Robillard, G.T.
1988-01-01
The cysteine of the membrane-bound mannitol-specific enzyme II (EII/sup Mtl/) of the Escherichia coli phosphoenolpyruvate-dependent phosphotransferase system have been labeled with 4-vinylpyridine. After proteolytic breakdown and reversed-phase HPLC, the peptides containing cysteines 110, 384, and 571 could be identified. N-Ethylmaleimide (NEM) treatment of the native unphosphorylated enzyme results in incorporation of one NEM label per molecule and loss of enzymatic activity. NEM treatment and inactivation prevented 4-vinylpyridine incorporation into the Cys-384-containing peptide, identifying this residue as the activity-linked cysteine. Both oxidation and phosphorylation of the native enzyme protected the enzyme against NEM labeling of Cys-384. Positive identification of the activity-linked cysteine was accomplished by inactivation with [ 14 C]iodoacetamide, proteolytic fragmentation, isolation of the peptide, and amino acid sequencing
Assmann, Alexander; Struß, Marc; Schiffer, Franziska; Heidelberg, Friederike; Munakata, Hiroshi; Timchenko, Elena V; Timchenko, Pavel E; Kaufmann, Tim; Huynh, Khon; Sugimura, Yukiharu; Leidl, Quentin; Pinto, Antonio; Stoldt, Volker R; Lichtenberg, Artur; Akhyari, Payam
2017-12-01
Low immunogenicity and high repopulation capacity are crucial determinants for the functional and structural performance of acellular cardiovascular implants. The present study evaluates a detergent-free, non-proteolytic, actin-disassembling regimen (BIO) for decellularization of heart valve and vessel grafts, particularly focusing on their bio-functionality. Rat aortic conduits (rAoC; n = 89) and porcine aortic valve samples (n = 106) are decellularized using detergents (group DET) or the BIO regimen. BIO decellularization results in effective elimination of cellular proteins and significantly improves removal of DNA as compared with group DET, while the extracellular matrix (ECM) structure as well as mechanical properties are preserved. The architecture of rAoC in group BIO allows for improved bio-functionalization with fibronectin (FN) in a standardized rat implantation model: BIO treatment significantly increases speed and amount of autologous medial cellular repopulation in vivo (p < 0.001) and decreases the formation of hyperplastic intima (p < 0.001) as compared with FN-coated DET-decellularized grafts. Moreover, there are no signs of infiltration with inflammatory cells. The present biological, detergent-free, non-proteolytic regimen balances effective decellularization and ECM preservation in cardiovascular grafts, and provides optimized bio-functionality. Additionally, this study implies that the actin-disassembling regimen may be a promising approach for bioengineering of acellular scaffolds from other muscular tissues, as for example myocardium or intestine. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.
Directory of Open Access Journals (Sweden)
Nur Maulida Safitri
2017-08-01
Full Text Available The aim of this study is to isolate the antioxidative peptide from Spirulina platensis. Peptide was obtained by proteolytic digestion, ultrafiltration, fractionation by RP-HPLC, identified by LC-MS/MS—MASCOT Distiller and measured its antioxidant activity by DPPH (2.2-Diphenyl-1-picrylhydrazyl assay. Results showed that thermolysin was the most effective enzyme to digest this algae. The active peptide Phe-Ser-Glu-Ser-Ser-Ala-Pro-Glu-Gln-His-Tyr (m/z 1281.51 was identified and synthetized, which exhibited 45.98 ± 1.7% at concentration 128.15 µg/mL. Therefore, S. platensis is indicated as a potential therapeutic source for combating oxidative stress.
Measurement of the activity of electron capturing isotopes
International Nuclear Information System (INIS)
Szoerenyi, A.
1980-01-01
In order to measure precisely the activity of electron capturing isotopes, an equipment was constructed for the detection the X-photons, the Auger- and the conversing electrons by a high-pressure, gas-flow 4π proportional counter. The proportional counter and the NaI(Tl) scintillation counter are placed in a common lead-shielding, thus, the equipment is suited for the measurement of radioisotopes decaying in coincidence. The structure of the proportional counter and of the pressure-control system are detailed. As an example, the energy spectra of a 109 Cd solution, taken at different pressures, are published. At a pressure of 1.1 MPa the 3 peaks are well separated. The results of an international test, in which the radioactivity of a 57 Co sample was determined, are published, too. (L.E.)
Active pixel sensor array with electronic shuttering
Fossum, Eric R. (Inventor)
2002-01-01
An active pixel cell includes electronic shuttering capability. The cell can be shuttered to prevent additional charge accumulation. One mode transfers the current charge to a storage node that is blocked against accumulation of optical radiation. The charge is sampled from a floating node. Since the charge is stored, the node can be sampled at the beginning and the end of every cycle. Another aspect allows charge to spill out of the well whenever the charge amount gets higher than some amount, thereby providing anti blooming.
Extraction of an urease-active organo-complex from soil.
Burns, R. G.; El-Sayed, M. H.; Mclaren, A. D.
1972-01-01
Description of an extraction from a Dublin clay loam soil of a colloidal organic matter complex that is urease active and, by X-ray analysis, free of clays. Urease activity in the clay-free precipitates, as in the soil, was not destroyed by the activity of an added proteolytic enzyme, pronase. This is attributed to the circumstance that native soil urease resides in organic colloidal particles with pores large enough for water, urea, ammonia, and carbon dioxide to pass freely, but nevertheless small enough to exclude pronase.
Effect of amino acid substitution on biological activity of cyanophlyctin-β and brevinin-2R
Ghorani-Azam, Adel; Balali-Mood, Mahdi; Aryan, Ehsan; Karimi, Gholamreza; Riahi-Zanjani, Bamdad
2018-04-01
Antimicrobial peptides (AMPs), as ancient immune components, are found in almost all types of living organisms. They are bioactive components with strong antibacterial, antiviral, and anti-tumor properties. In this study, we designed three sequences of antimicrobial peptides to study the effects of structural changes in biological activity compared with original peptides, cyanophlyctin β, and brevinin-2R. For antibacterial activity, two Gram-positive (Staphylococcus aureus and S. epidermidis) and two Gram-negative bacteria (Escherichia coli and Pseudomonas aeroginosa) were assayed. Unlike cyanophlyctin β and brevinin-2R, the synthesized peptide (brevinin-M1, brevinin-M2 and brevinin-M3) showed no considerable antibacterial properties. Hemolytic activity of these peptides was also ignorable even at very high concentrations of 2 mg/ml. However, after proteolytic digestion by trypsin, the peptides showed antibacterial activity comparable to their original template sequences. Structural prediction suggested that the motif sequence responsible for antibacterial activity may be re-exposed to bacterial cell membrane after proteolytic digestion. Also, findings showed that only a small change in primary sequence and therefore structure of peptides may result in a significant alteration in biological activity.
New intracellular activities of matrix metalloproteinases shine in the moonlight.
Jobin, Parker G; Butler, Georgina S; Overall, Christopher M
2017-11-01
Adaption of a single protein to perform multiple independent functions facilitates functional plasticity of the proteome allowing a limited number of protein-coding genes to perform a multitude of cellular processes. Multifunctionality is achievable by post-translational modifications and by modulating subcellular localization. Matrix metalloproteinases (MMPs), classically viewed as degraders of the extracellular matrix (ECM) responsible for matrix protein turnover, are more recently recognized as regulators of a range of extracellular bioactive molecules including chemokines, cytokines, and their binders. However, growing evidence has convincingly identified select MMPs in intracellular compartments with unexpected physiological and pathological roles. Intracellular MMPs have both proteolytic and non-proteolytic functions, including signal transduction and transcription factor activity thereby challenging their traditional designation as extracellular proteases. This review highlights current knowledge of subcellular location and activity of these "moonlighting" MMPs. Intracellular roles herald a new era of MMP research, rejuvenating interest in targeting these proteases in therapeutic strategies. This article is part of a Special Issue entitled: Matrix Metalloproteinases edited by Rafael Fridman. Copyright © 2017 Elsevier B.V. All rights reserved.
Single-active-electron potentials for molecules in intense laser fields
DEFF Research Database (Denmark)
Abu-Samha, Mahmoud; Madsen, Lars Bojer
2010-01-01
Single-active-electron potentials are computed for selected molecules, and molecular wave functions with the correct asymptotic behavior are produced. Asymptotic expansion coefficients are extracted from the wave functions and used to compute alignment-dependent ionization yields from molecular...
Extracellular proteolytic activity of Deinococcus geothermalis ...
African Journals Online (AJOL)
Nonionic detergents like Triton X-100 and Tween 80 did not affect catalytic properties. It suggested that the enzyme produced by D. geothermalis could be used as a component of detergents. Keywords: Deinococcus geothermalis, alkaline protease, detergents, thermostability. African Journal of Biotechnology Vol. 12(25) ...
The relationship between ionospheric temperature, electron density and solar activity
International Nuclear Information System (INIS)
McDonald, J.N.; Williams, P.J.S.
1980-01-01
In studying the F-region of the ionosphere several authors have concluded that the difference between the electron temperature Tsub(e) and the ion temperature Tsub(i) is related to the electron density N. It was later noted that solar activity (S) was involved and an empirical relationship of the following form was established: Tsub(e)-Tsub(i) = A-BN+CS. The present paper extends this work using day-time data over a four year period. The results are given and discussed. A modified form of the empirical relation is proposed. (U.K.)
Bischak, Connor G; Hetherington, Craig L; Wang, Zhe; Precht, Jake T; Kaz, David M; Schlom, Darrell G; Ginsberg, Naomi S
2015-05-13
We demonstrate a new nanoimaging platform in which optical excitations generated by a low-energy electron beam in an ultrathin scintillator are used as a noninvasive, near-field optical scanning probe of an underlying sample. We obtain optical images of Al nanostructures with 46 nm resolution and validate the noninvasiveness of this approach by imaging a conjugated polymer film otherwise incompatible with electron microscopy due to electron-induced damage. The high resolution, speed, and noninvasiveness of this "cathodoluminescence-activated" platform also show promise for super-resolution bioimaging.
Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase
Energy Technology Data Exchange (ETDEWEB)
Sode, Olaseni; Voth, Gregory A., E-mail: gavoth@uchicago.edu [Department of Chemistry, James Franck Institute, Institute for Biophysical Dynamics, Computation Institute, The University of Chicago, Chicago, Illinois 60637, USA and Computing, Environment and Life Sciences, Argonne National Laboratory, Argonne, Illinois 60439 (United States)
2014-12-14
Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A{sup 2−}, a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A{sup 2-} by closing a cavity that could otherwise fill with water near the proximal Fe of the active site.
Electron transfer activation of a second water channel for proton transport in [FeFe]-hydrogenase
International Nuclear Information System (INIS)
Sode, Olaseni; Voth, Gregory A.
2014-01-01
Hydrogenase enzymes are important because they can reversibly catalyze the production of molecular hydrogen. Proton transport mechanisms have been previously studied in residue pathways that lead to the active site of the enzyme via residues Cys299 and Ser319. The importance of this pathway and these residues has been previously exhibited through site-specific mutations, which were shown to interrupt the enzyme activity. It has been shown recently that a separate water channel (WC2) is coupled with electron transport to the active site of the [FeFe]-hydrogenase. The water-mediated proton transport mechanisms of the enzyme in different electronic states have been studied using the multistate empirical valence bond reactive molecular dynamics method, in order to understand any role WC2 may have in facilitating the residue pathway in bringing an additional proton to the enzyme active site. In a single electronic state A 2− , a water wire was formed through which protons can be transported with a low free energy barrier. The remaining electronic states were shown, however, to be highly unfavorable to proton transport in WC2. A double amino acid substitution is predicted to obstruct proton transport in electronic state A 2- by closing a cavity that could otherwise fill with water near the proximal Fe of the active site
Sah, B N P; Vasiljevic, T; McKechnie, S; Donkor, O N
2014-08-01
Search for bioactive peptides is intensifying because of the risks associated with the use of synthetic therapeutics, thus peptide liberation by lactic acid bacteria and probiotics has received a great focus. However, proteolytic capacity of these bacteria is strain specific. The study was conducted to establish proteolytic activity of Lactobacillus acidophilus (ATCC® 4356™), Lactobacillus casei (ATCC® 393™) and Lactobacillus paracasei subsp. paracasei (ATCC® BAA52™) in yogurt. Crude peptides were separated by high-speed centrifugation and tested for antioxidant and antimutagenic activities. The degree of proteolysis highly correlated with these bioactivities, and its value (11.91%) for samples containing all the cultures was double that of the control. Liberated peptides showed high radical scavenging activities with 1,1-diphenyl-2-picrylhydrazyl and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), IC50 1.51 and 1.63mg/ml, respectively and strong antimutagenicity (26.35%). These probiotics enhanced the generation of bioactive peptides and could possibly be commercially applied in new products, or production of novel anticancer peptides. Crown Copyright © 2014. Published by Elsevier Ltd. All rights reserved.
Regeneration of used activated carbon by electron beam irradiation
International Nuclear Information System (INIS)
Arai, H.; Hosono, M.; Zhu, G.; Miyata, T.
1992-01-01
The adsorbing power of granular activated carbons which adsorbed sodium laurylsulfate were most effectively recovered by irradiation of high energy electron beams in nitrogen stream, and the carbon was hardly lost by irradiation. The regeneration was induced mainly by microscopic heating of adsorption sites. Regeneration was also confirmed by adsorption endotherms. Regeneration cost was tentatively evaluated. (author)
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.
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Holger B Kramer
2010-05-01
Full Text Available The earliest immune responses activated in acute human immunodeficiency virus type 1 infection (AHI exert a critical influence on subsequent virus spread or containment. During this time frame, components of the innate immune system such as macrophages and DCs, NK cells, beta-defensins, complement and other anti-microbial factors, which have all been implicated in modulating HIV infection, may play particularly important roles. A proteomics-based screen was performed on a cohort from whom samples were available at time points prior to the earliest positive HIV detection. The ability of selected factors found to be elevated in the plasma during AHI to inhibit HIV-1 replication was analyzed using in vitro PBMC and DC infection models. Analysis of unique plasma donor panels spanning the eclipse and viral expansion phases revealed very early alterations in plasma proteins in AHI. Induction of acute phase protein serum amyloid A (A-SAA occurred as early as 5-7 days prior to the first detection of plasma viral RNA, considerably prior to any elevation in systemic cytokine levels. Furthermore, a proteolytic fragment of alpha-1-antitrypsin (AAT, termed virus inhibitory peptide (VIRIP, was observed in plasma coincident with viremia. Both A-SAA and VIRIP have anti-viral activity in vitro and quantitation of their plasma levels indicated that circulating concentrations are likely to be within the range of their inhibitory activity. Our results provide evidence for a first wave of host anti-viral defense occurring in the eclipse phase of AHI prior to systemic activation of other immune responses. Insights gained into the mechanism of action of acute-phase reactants and other innate molecules against HIV and how they are induced could be exploited for the future development of more efficient prophylactic vaccine strategies.
Petersen, Lauren M; Tisa, Louis S
2014-11-01
A newly recognized Serratia species, termed South African Caenorhabditis briggsae isolate (SCBI), is both a mutualist of the nematode Caenorhabditis briggsae KT0001 and a pathogen of lepidopteran insects. Serratia sp. strain SCBI displays high proteolytic activity, and because secreted proteases are known virulence factors for many pathogens, the purpose of this study was to identify genes essential for extracellular protease activity in Serratia sp. strain SCBI and to determine what role proteases play in insect pathogenesis and cytotoxicity. A bank of 2,100 transposon mutants was generated, and six SCBI mutants with defective proteolytic activity were identified. These mutants were also defective in cytotoxicity. The mutants were found defective in genes encoding the following proteins: alkaline metalloprotease secretion protein AprE, a BglB family transcriptional antiterminator, an inosine/xanthosine triphosphatase, GidA, a methyl-accepting chemotaxis protein, and a PIN domain protein. Gene expression analysis on these six mutants showed significant downregulation in mRNA levels of several different types of predicted protease genes. In addition, transcriptome sequencing (RNA-seq) analysis provided insight into how inactivation of AprE, GidA, and a PIN domain protein influences motility and virulence, as well as protease activity. Using quantitative reverse transcription-PCR (qRT-PCR) to further characterize expression of predicted protease genes in wild-type Serratia sp. SCBI, the highest mRNA levels for the alkaline metalloprotease genes (termed prtA1 to prtA4) occurred following the death of an insect host, while two serine protease and two metalloprotease genes had their highest mRNA levels during active infection. Overall, these results indicate that proteolytic activity is essential for cytotoxicity in Serratia sp. SCBI and that its regulation appears to be highly complex. Copyright © 2014, American Society for Microbiology. All Rights Reserved.
International Nuclear Information System (INIS)
Martin, Thomas; Uhder, Kerstin; Kurek, Ralf; Roeddiger, Sandra; Schneider, Lida; Vogt, Hans-Georg; Heyd, Reinhard; Zamboglou, Nikolaos
2002-01-01
Purpose: Does prophylactic treatment with proteolytic enzymes reduce acute toxicity of adjuvant pelvic radiotherapy? Material and methods: Fifty-six patients with an indication for adjuvant pelvic irradiation after curative surgery were double-blind randomized. All patients took 3x4 capsules study medication daily during radiotherapy. Twenty-eight patients in the enzyme group (EG) received capsules containing papain, trypsin and chymotrypsin, 28 in the placebo group (PG) received placebo capsules. All patients were irradiated with 5x1.8 Gy weekly to 50.4 Gy using four-field-box technique after CT-based planning. Primary objective was the grade of diarrhea, nausea, vomiting, fatigue and epitheliolysis during radiotherapy. Secondary objectives were the number of supportive medications and treatment interruptions due to acute toxicity. Results: None/mild diarrhea: 43% EG, 64% PG. Moderate/severe diarrhea: 57% EG, 36% PG (P=0.11). Mean duration: 11 days in EG, 10 days in PG. None/mild nausea: 93% EG, 93% PG. Moderate/severe nausea: 7% EG, 7% PG. None/mild vomiting: 100% EG, 97% PG. None/mild fatigue: 82% EG, 93% PG. Moderate/severe fatigue: 18% EG, 7% PG (P=0.23). None/mild epitheliolysis: 75% EG, 93% PG. Moderate/severe epitheliolysis: 25% EG, 7% PG (P=0.16). Treatment interruption (mean days): 2.44 in EG, 1.46 in PG. Number of supportive medication: 29 in EG, 19 in PG. Conclusions: The prophylactic use of proteolytic enzymes does not reduce acute toxicities, treatment interruptions and number of supportive medication and therefore does not improve tolerance of adjuvant pelvic radiotherapy
Directory of Open Access Journals (Sweden)
Timothy F Meiller
Full Text Available Oropharyngeal candidiasis is an opportunistic infection considered to be a harbinger of AIDS. The etiologic agent Candida albicans is a fungal species commonly colonizing human mucosal surfaces. However, under conditions of immune dysfunction, colonizing C. albicans can become an opportunistic pathogen causing superficial or even life-threatening infections. The reasons behind this transition, however, are not clear. In the oral cavity, salivary antimicrobial peptides are considered to be an important part of the host innate defense system in the prevention of microbial colonization. Histatin-5 specifically has exhibited potent activity against C. albicans. Our previous studies have shown histatin-5 levels to be significantly reduced in the saliva of HIV+ individuals, indicating an important role for histatin-5 in keeping C. albicans in its commensal stage. The versatility in the pathogenic potential of C. albicans is the result of its ability to adapt through the regulation of virulence determinants, most notably of which are proteolytic enzymes (Saps, involved in tissue degradation. In this study, we show that C. albicans cells efficiently and rapidly degrade histatin-5, resulting in loss of its anti-candidal potency. In addition, we demonstrate that this cellular activity is due to proteolysis by a member of the secreted aspartic proteases (Sap family involved in C. albicans pathogenesis. Specifically, the proteolysis was attributed to Sap9, in turn identifying histatin-5 as the first host-specific substrate for that isoenzyme. These findings demonstrate for the first time the ability of a specific C. albicans enzyme to degrade and deactivate a host antimicrobial peptide involved in the protection of the oral mucosa against C. albicans, thereby providing new insights into the factors directing the transition of C. albicans from commensal to pathogen, with important clinical implications for alternative therapy. This report characterizes the
International Nuclear Information System (INIS)
Grossman, A.
1984-01-01
N-benzoyl-L-argininyl-p-nitroanilide (BAN), a synthetic substrate for trypsin-like proteolytic enzymes, is a potent activator of [ 3 H]estradiol-binding to a protein present in rat pancreas. When partially purified, this protein is almost devoid of [ 3 H]estradiol-binding activity in the absence of an endogenous accessory factor. BAN can mimic the natural coligand in this steroid binding reaction. The effect of BAN is specific since a number of derivatives of this substance are inactive or may even inhibit steroid binding. It is unlikely that BAN exerts this stimulatory action indirectly, possibly by preventing proteolytic inactivation of the [ 3 H]estradiol-binding protein, since preincubation of the protein in the absence of BAN resulted neither in reduced rate, nor extent, of steroid binding following BAN addition. Also, a number of protease inhibitors had no effect on the binding reaction. Of those inhibitors tested, only antipain significantly enhanced binding of [ 3 H]estradiol, but only about 20 percent as effectively as BAN. 13 references, 1 figure, 2 tables
Sit, Cindy H P; Lam, Jessica W K; McKenzie, Thomas L
2010-07-01
Interactive electronic games have recently been popularized and are believed to help promote children's physical activity (PA). The purpose of the study was to examine preferences and PA levels during interactive and online electronic games among overweight and nonoverweight boys and girls. Using a modification of the SOFIT, we systematically observed 70 Hong Kong Chinese children (35 boys, 35 girls; 50 nonoverweight, 20 overweight), age 9 to 12 years, during 2 60-minute recreation sessions and recorded their game mode choices and PA levels. During Session One children could play either an interactive or an online electronic bowling game and during Session Two they could play an interactive or an online electronic running game. Children chose to play the games during 94% of session time and split this time between interactive (52%) and online (48%) versions. They engaged in significantly more moderate-to-vigorous physical activity (MVPA) during interactive games than their online electronic versions (70% vs. 2% of game time). Boys and nonoverweight children expended relatively more energy during the interactive games than girls and overweight children, respectively. New-generation interactive games can facilitate physical activity in children, and given the opportunity children may select them over sedentary versions.
Proteolytic activity of beef liver determined by natural /sup 125/I-labelled substrates
Energy Technology Data Exchange (ETDEWEB)
Blahovec, J; Ondrus, I [Vysoka Skola Veterinarska, Kosice (Czechoslovakia)
1975-07-01
The method of determining the enzymatic activity of acid proteinases is described. The method is based on the use of /sup 125/I-labelled natural protein substrates. /sup 125/I-labelled albumin, /sup 125/I-globulin and /sup 125/I-insulin were tested for the determination of activities. All the substrates were hydrolyzed with the enzymes of the supernatant fraction (106,000 g) of beef liver homogenate in the acid pH zone. Optimum enzymatic reaction conditions were tested, the dependence of the reaction on the enzyme concentration, time and temperature was determined, pH optimum was ascertained for the individual substrates, and pH stability was determined. The results show that the method is suitable for determining the enzymatic activity of proteinases of cathepsin character.
COMPARATIVE STUDIES OF THREE METHODS FOR MEASURING PEPSIN ACTIVITY
Loken, Merle K.; Terrill, Kathleen D.; Marvin, James F.; Mosser, Donn G.
1958-01-01
Comparison has been made of a simple method originated by Absolon and modified in our laboratories for assay of proteolytic activity using RISA (radioactive iodinated serum albumin—Abbott Laboratories), with the commonly used photometric methods of Anson and Kunitz. In this method, pepsin was incubated with an albumin substrate containing RISA, followed by precipitation of the undigested substrate with trichloroacetic acid and measurement of radioactive digestion products in the supernatant fluid. The I131—albumin bond was shown in the present studies to be altered only by the proteolytic activity, and not by the incubation procedures at various values of pH. Any free iodine present originally in the RISA was removed by a single passage through a resin column (amberlite IRA-400-C1). Pepsin was shown to be most stable in solution at a pH of 5.5. Activity of pepsin was shown to be maximal when it was incubated with albumin at a pH of 2.5. Pepsin activity was shown to be altered in the presence of various electrolytes. Pepsin activity measured by the RISA and Anson methods as a function of concentration or of time of incubation indicated that these two methods are in good agreement and are equally sensitive. Consistently smaller standard errors were obtained by the RISA method of pepsin assay than were obtained with either of the other methods. PMID:13587910
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Roman Szabo
Full Text Available Loss of either hepatocyte growth factor activator inhibitor (HAI-1 or -2 is associated with embryonic lethality in mice, which can be rescued by the simultaneous inactivation of the membrane-anchored serine protease, matriptase, thereby demonstrating that a matriptase-dependent proteolytic pathway is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase targets, and screening for the rescue of embryonic development. Hypomorphic mutations in Prss8, encoding the GPI-anchored serine protease, prostasin (CAP1, PRSS8, restored placentation and normal development of HAI-1-deficient embryos and prevented early embryonic lethality, mid-gestation lethality due to placental labyrinth failure, and neural tube defects in HAI-2-deficient embryos. Inactivation of genes encoding c-Met, protease-activated receptor-2 (PAR-2, or the epithelial sodium channel (ENaC alpha subunit all failed to rescue embryonic lethality, suggesting that deregulated matriptase-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during neural tube closure. Paradoxically, although matriptase auto-activates and is a well-established upstream epidermal activator of prostasin, biochemical analysis of matriptase- and prostasin-deficient placental tissues revealed a requirement of prostasin for conversion of the matriptase zymogen to active matriptase, whereas prostasin zymogen activation was matriptase-independent.
Prajapati, Darshna B.; Kapadiya, Dharti B.; Jain, Amit Kumar; Mehta, Bhavbhuti M.; Darji, Vijaykumar B.; Aparnathi, Kishorkumar D.
2017-01-01
Aim: The study was undertaken to find out the physicochemical characteristics, selected processing-related parameters and activity of selected enzymes in Surti goat milk. Materials and Methods: Milk samples from Surti goats and buffalo milk samples were collected during the period from July 2013 to January 2014 at Reproductive Biology Research Unit, Anand Agricultural University (AAU), Anand. Milk samples from Kankrej cows were collected from Livestock Research Station, AAU, Anand. Samples were analyzed for physicochemical characteristics such as acidity, viscosity, surface tension, specific gravity, refractive index, freezing point, and electrical conductivity. Samples were also analyzed for selected processing-related parameters such as heat coagulation time (HCT), rennet coagulation time (RCT), rate of acid production by starter culture, alcohol stability, and activity of selected enzymes such as alkaline phosphatase activity, catalase activity, proteolytic activity, and lipase activity. Results: Goat milk had the highest acidity, viscosity and surface tension, followed by cow milk and buffalo milk. However, the differences in acidity, specific gravity, surface tension, refractive index, electrical conductivity, HCT and lipase activity of three types of milk studied, viz., goat, cow, and buffalo milk were found statistically non-significant (pmilk had the highest specific gravity, followed by those found in cow and goat milk. The viscosity, freezing point and RCT of goat milk was significantly lower (p>0.05) than that of the buffalo milk. However, the difference in viscosity, freezing point and RCT of goat milk and that of the cow milk was statistically non-significant. The cow milk had the highest refractive index, followed by goat and buffalo milk. The cow milk had the highest proteolytic activity and heat coagulation time (HCT), followed by those found in buffalo and goat milk. The goat milk had the lowest freezing point, lipase activity, and RCT, followed by
Directory of Open Access Journals (Sweden)
Darshna B. Prajapati
2017-05-01
Full Text Available Aim: The study was undertaken to find out the physicochemical characteristics, selected processing-related parameters and activity of selected enzymes in Surti goat milk. Materials and Methods: Milk samples from Surti goats and buffalo milk samples were collected during the period from July 2013 to January 2014 at Reproductive Biology Research Unit, Anand Agricultural University (AAU, Anand. Milk samples from Kankrej cows were collected from Livestock Research Station, AAU, Anand. Samples were analyzed for physicochemical characteristics such as acidity, viscosity, surface tension, specific gravity, refractive index, freezing point, and electrical conductivity. Samples were also analyzed for selected processing-related parameters such as heat coagulation time (HCT, rennet coagulation time (RCT, rate of acid production by starter culture, alcohol stability, and activity of selected enzymes such as alkaline phosphatase activity, catalase activity, proteolytic activity, and lipase activity. Results: Goat milk had the highest acidity, viscosity and surface tension, followed by cow milk and buffalo milk. However, the differences in acidity, specific gravity, surface tension, refractive index, electrical conductivity, HCT and lipase activity of three types of milk studied, viz., goat, cow, and buffalo milk were found statistically non-significant (p0.05 than that of the buffalo milk. However, the difference in viscosity, freezing point and RCT of goat milk and that of the cow milk was statistically non-significant. The cow milk had the highest refractive index, followed by goat and buffalo milk. The cow milk had the highest proteolytic activity and heat coagulation time (HCT, followed by those found in buffalo and goat milk. The goat milk had the lowest freezing point, lipase activity, and RCT, followed by those found in cow and buffalo milk. The goat milk had the highest electrical conductivity, followed by those found in buffalo and cow milk. The
2011-07-08
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Light-Enhanced Antibacterial Activity of Graphene Oxide, Mainly via Accelerated Electron Transfer.
Chong, Yu; Ge, Cuicui; Fang, Ge; Wu, Renfei; Zhang, He; Chai, Zhifang; Chen, Chunying; Yin, Jun-Jie
2017-09-05
Before graphene derivatives can be exploited as next-generation antimicrobials, we must understand their behavior under environmental conditions. Here, we demonstrate how exposure to simulated sunlight significantly enhances the antibacterial activity of graphene oxide (GO) and reveal the underlying mechanism. Our measurements of reactive oxygen species (ROS) showed that only singlet oxygen ( 1 O 2 ) is generated by GO exposed to simulated sunlight, which contributes only slightly to the oxidation of antioxidant biomolecules. Unexpectedly, we find the main cause of oxidation is light-induced electron-hole pairs generated on the surface of GO. These light-induced electrons promote the reduction of GO, introducing additional carbon-centered free radicals that may also enhance the antibacterial activities of GO. We conclude that GO-mediated oxidative stress mainly is ROS-independent; simulated sunlight accelerates the transfer of electrons from antioxidant biomolecules to GO, thereby destroying bacterial antioxidant systems and causing the reduction of GO. Our insights will help support the development of graphene for antibacterial applications.
Loprinzi, Paul D; Schary, David P; Cardinal, Bradley J
2013-01-01
The primary purpose of the present study was to examine adherence to current active play and electronic media use guidelines in a sample of US preschool-aged children and to examine whether differences occurred across gender and parental education. 164 parents completed an on-line survey to estimate preschool children's active play and sedentary behaviors. For weekdays, 50% of the sample met the active play guideline with this increasing to 65% during the weekend. With respect to electronic media use, 90% of the sample met guidelines during the week, with the percentage meeting guidelines dropping to 78% during the weekend. A greater percentage of preschool children from high parental education families (83.4 ± 3.3) met electronic media use guidelines on the weekends, compared to preschool children from low parental education families (59.4 ± 8.1) (p = 0.002). Our findings indicate that a substantial portion of preschool children are not meeting active play guidelines and that adherence to active play and electronic media use guidelines may be influenced by parental education.
Patterson, Rachel A.; Juarez, Michelle T.; Hermann, Anita; Sasik, Roman; Hardiman, Gary; McGinnis, William
2013-01-01
After injury to the animal epidermis, a variety of genes are transcriptionally activated in nearby cells to regenerate the missing cells and facilitate barrier repair. The range and types of diffusible wound signals that are produced by damaged epidermis and function to activate repair genes during epidermal regeneration remains a subject of very active study in many animals. In Drosophila embryos, we have discovered that serine protease function is locally activated around wound sites, and is also required for localized activation of epidermal repair genes. The serine protease trypsin is sufficient to induce a striking global epidermal wound response without inflicting cell death or compromising the integrity of the epithelial barrier. We developed a trypsin wounding treatment as an amplification tool to more fully understand the changes in the Drosophila transcriptome that occur after epidermal injury. By comparing our array results with similar results on mammalian skin wounding we can see which evolutionarily conserved pathways are activated after epidermal wounding in very diverse animals. Our innovative serine protease-mediated wounding protocol allowed us to identify 8 additional genes that are activated in epidermal cells in the immediate vicinity of puncture wounds, and the functions of many of these genes suggest novel genetic pathways that may control epidermal wound repair. Additionally, our data augments the evidence that clean puncture wounding can mount a powerful innate immune transcriptional response, with different innate immune genes being activated in an interesting variety of ways. These include puncture-induced activation only in epidermal cells in the immediate vicinity of wounds, or in all epidermal cells, or specifically in the fat body, or in multiple tissues. PMID:23637905
Fermentation of sugar to ethyl alcohol in the presence of proteolytic enzymes
Energy Technology Data Exchange (ETDEWEB)
Coates, E W; Conde Julio, C
1963-06-11
Sugar is fermented to EtOH by yeasts capable of elaborating zymase and proteolytic enzymes, the zymase component comprising exceptionally large amounts of phosphatase. Saccharomyces ellipsoideus was acclimated to 20% EtOH by growing on fresh pineapple juice in a medium consisting of malt sirup 15, sugar sirup 3, and pineapple juice 82%. An aqueous solution of 2000 gallons of sugar cane molasses in H/sub 2/O to give a Brix of 16/sup 0/ was placed in a 48,000-gallon fermentor. S. ellipsoideus with a cell constant of 1 x 10/sup 9/ cells/ml was added, with sufficient H/sub 2/SO/sub 4/ to adjust the pH to approximately 4.5. Fermentation was carried out at 35/sup 0/ until the Brix dropped to 8/sup 0/, after which it was brought back to 16/sup 0/ by adding 6000 gallons of sirup containing nutrients in H/sub 2/O. This process was repeated with another 6000 and then 2000 gallons of sirup. The total fermentation required 48 h and the EtOH content was 15.25% by volume. Te EtOH was recovered in the usual manner by removal of solids and fractional distillation.
Serine proteases as candidates for proteolytic processing of angiotensin-I converting enzyme.
Aragão, Danielle S; de Andrade, Maria Claudina C; Ebihara, Fabiana; Watanabe, Ingrid K M; Magalhães, Dayane C B P; Juliano, Maria Aparecida; Hirata, Izaura Yoshico; Casarini, Dulce Elena
2015-01-01
Somatic angiotensin-I converting enzyme (sACE) is a broadly distributed peptidase which plays a role in blood pressure and electrolyte homeostasis by the conversion of angiotensin I into angiotensin II. N-domain isoforms (nACE) with 65 and 90 kDa have been described in body fluids, tissues and mesangial cells (MC), and a 90 kDa nACE has been described only in spontaneously hypertensive rats. The aim of this study was to investigate the existence of proteolytic enzymes that may act in the hydrolysis of sACE generating nACEs in MC. After the confirmation of the presence of ACE sheddases in Immortalized MC (IMC), we purified and characterized these enzymes using fluorogenic substrates specifically designed for ACE sheddases. Purified enzyme identified as a serine protease by N-terminal sequence was able to generate nACE. In the present study, we described for the first time the presence of ACE sheddases in IMC, identified as serine proteases able to hydrolyze sACE in vitro. Further investigations are necessary to elucidate the mechanisms responsible for the expression and regulation of ACE sheddases in MC and their roles in the generation of nACEs, especially the 90 kDa form possibly related to hypertension. Copyright © 2014 Elsevier B.V. All rights reserved.
Stachowicz, Jadwiga; Krajewska-Kułak, Elżbieta; Łukaszuk, Cecylia; Niewiadomy, A.
2014-01-01
Due to the increasing demand for new pharmaceuticals showing biological activity against pathogenic microorganisms, there is increasing search for new compounds with predicted biological activity. Variously substituted thioamide derivatives with 1.3 and 1.2 ring of thiazole and 1,3,4-thiadiazole, as well as pyrazole were assessed for their activity against Candida albicans. Activity of majority of tested thioamides was larger as compared with that of the reference drugs. The electron parameters of obtained N-heterocyclic thioamides were determined and dependencies on their biological activity against Candida albicans were studied. The best electron compliance of produced bindings with the activity against Candida albicans was observed for the derivatives containing 1,3,4-thiadiazole ring. PMID:25284926
Zhang, K.; Li, X.; Schiller, Q.; Gerhardt, D.; Zhao, H.; Millan, R.
2017-08-01
Earth's outer radiation belt electrons are highly dynamic. We study the detailed characteristics of relativistic electrons in the outer belt using measurements from the Colorado Student Space Weather Experiment (CSSWE) mission, a low Earth orbit (LEO) CubeSat, which traverses the radiation belt four times in one orbit ( 1.5 h) and has the advantage of measuring the dynamic activities of the electrons including their rapid precipitation. We focus on the measured electron response to geomagnetic activity for different energies to show that there are abundant sub-MeV electrons in the inner belt and slot region. These electrons are further enhanced during active times, while there is a lack of >1.63 MeV electrons in these regions. We also show that the variation of measured electron flux at LEO is strongly dependent on the local magnetic field strength, which is far from a dipole approximation. Moreover, a specific precipitation band, which happened on 19 January 2013, is investigated based on the conjunctive measurement of CSSWE, the Balloon Array for Radiation belt Relativistic Electron Losses, and one of the Polar Operational Environmental Satellites. In this precipitation band event, the net loss of the 0.58-1.63 MeV electrons (L = 3.5-6) is estimated to account for 6.8% of the total electron content.
Site selection of active damper for stabilizing power electronics based power distribution system
DEFF Research Database (Denmark)
Yoon, Changwoo; Wang, Xiongfei; Bak, Claus Leth
2015-01-01
electronics based power device, which provides an adjustable damping capability to the power system where the voltage harmonic instability is measured. It can stabilize by adjusting the equivalent node impedance with its plug and play feature. This feature gives many degrees of freedom of its installation......Stability in the nowadays distribution power system is endangered by interaction problems that may arise from newly added power-electronics based power devices. Recently, a new concept to deal with this higher frequency instability, the active damper, has been proposed. The active damper is a power...... point when the system has many nodes. Therefore, this paper addresses the proper placement of an active damper in an unstable small-scale power distribution system. A time-domain model of the Cigre benchmark low-vltage network is used as a test field. The result shows the active damper location...
International Nuclear Information System (INIS)
Santos, Elizabeth; Schmickler, Wolfgang
2008-01-01
A unified model for electrochemical electron transfer reactions which explicitly accounts for the electronic structure of the electrode recently proposed by us is applied to the hydrogen oxidation reaction at different metal electrocatalysts. We focus on the changes produced in the transition state (saddle point) as a consequence of the interactions with d-bands. We discuss different empirical correlations between properties of the metal and catalytic activity proposed in the past. We show which role is played by the band structure of the different metals and its interaction with the molecule for decreasing the activation barrier. Finally, we demonstrate why some metals are better electrocatalysts for the hydrogen electro-oxidation reaction than others
DEFF Research Database (Denmark)
Szabo, Roman; Uzzun Sales, Katiuchia; Kosa, Peter
2012-01-01
is a critical developmental target for both protease inhibitors. Here, we performed a genetic epistasis analysis to identify additional components of this pathway by generating mice with combined deficiency in either HAI-1 or HAI-2, along with genes encoding developmentally co-expressed candidate matriptase......-prostasin activity causes developmental failure independent of aberrant c-Met and PAR-2 signaling or impaired epithelial sodium transport. Furthermore, phenotypic analysis of PAR-1 and matriptase double-deficient embryos suggests that the protease may not be critical for focal proteolytic activation of PAR-2 during...
Directory of Open Access Journals (Sweden)
Yumiko Obayashi
2017-10-01
Full Text Available Microbial extracellular hydrolytic enzymes that degrade organic matter in aquatic ecosystems play key roles in the biogeochemical carbon cycle. To provide linkages between hydrolytic enzyme activities and genomic or metabolomic studies in aquatic environments, reliable measurements are required for many samples at one time. Extracellular proteases are one of the most important classes of enzymes in aquatic microbial ecosystems, and protease activities in seawater are commonly measured using fluorogenic model substrates. Here, we examined several concerns for measurements of extracellular protease activities (aminopeptidases, and trypsin-type, and chymotrypsin-type activities in seawater. Using a fluorometric microplate reader with low protein binding, 96-well microplates produced reliable enzymatic activity readings, while use of regular polystyrene microplates produced readings that showed significant underestimation, especially for trypsin-type proteases. From the results of kinetic experiments, this underestimation was thought to be attributable to the adsorption of both enzymes and substrates onto the microplate. We also examined solvent type and concentration in the working solution of oligopeptide-analog fluorogenic substrates using dimethyl sulfoxide (DMSO and 2-methoxyethanol (MTXE. The results showed that both 2% (final concentration of solvent in the mixture of seawater sample and substrate working solution DMSO and 2% MTXE provide similarly reliable data for most of the tested substrates, except for some substrates which did not dissolve completely in these assay conditions. Sample containers are also important to maintain the level of enzyme activity in natural seawater samples. In a small polypropylene containers (e.g., standard 50-mL centrifugal tube, protease activities in seawater sample rapidly decreased, and it caused underestimation of natural activities, especially for trypsin-type and chymotrypsin-type proteases. In
DEFF Research Database (Denmark)
Cornelia Peeters, Miriam; Fokkelman, Michiel; Boogaard, Bob
2015-01-01
Adhesion G protein-coupled receptors (ADGRs) are believed to be activated by auto-proteolytic cleavage of their very large extracellular N-terminal domains normally acting as a negative regulator of the intrinsically constitutively active seven transmembrane domain. ADGRG2 (or GPR64) which...
Pettit, S C; Moody, M D; Wehbie, R S; Kaplan, A H; Nantermet, P V; Klein, C A; Swanstrom, R
1994-01-01
The proteolytic processing sites of the human immunodeficiency virus type 1 (HIV-1) Gag precursor are cleaved in a sequential manner by the viral protease. We investigated the factors that regulate sequential processing. When full-length Gag protein was digested with recombinant HIV-1 protease in vitro, four of the five major processing sites in Gag were cleaved at rates that differ by as much as 400-fold. Three of these four processing sites were cleaved independently of the others. The CA/p...
Lyons, Elizabeth J; Lewis, Zakkoyya H; Mayrsohn, Brian G; Rowland, Jennifer L
2014-08-15
Electronic activity monitors (such as those manufactured by Fitbit, Jawbone, and Nike) improve on standard pedometers by providing automated feedback and interactive behavior change tools via mobile device or personal computer. These monitors are commercially popular and show promise for use in public health interventions. However, little is known about the content of their feedback applications and how individual monitors may differ from one another. The purpose of this study was to describe the behavior change techniques implemented in commercially available electronic activity monitors. Electronic activity monitors (N=13) were systematically identified and tested by 3 trained coders for at least 1 week each. All monitors measured lifestyle physical activity and provided feedback via an app (computer or mobile). Coding was based on a hierarchical list of 93 behavior change techniques. Further coding of potentially effective techniques and adherence to theory-based recommendations were based on findings from meta-analyses and meta-regressions in the research literature. All monitors provided tools for self-monitoring, feedback, and environmental change by definition. The next most prevalent techniques (13 out of 13 monitors) were goal-setting and emphasizing discrepancy between current and goal behavior. Review of behavioral goals, social support, social comparison, prompts/cues, rewards, and a focus on past success were found in more than half of the systems. The monitors included a range of 5-10 of 14 total techniques identified from the research literature as potentially effective. Most of the monitors included goal-setting, self-monitoring, and feedback content that closely matched recommendations from social cognitive theory. Electronic activity monitors contain a wide range of behavior change techniques typically used in clinical behavioral interventions. Thus, the monitors may represent a medium by which these interventions could be translated for
Soft Active Materials for Actuation, Sensing, and Electronics
Kramer, Rebecca Krone
Future generations of robots, electronics, and assistive medical devices will include systems that are soft and elastically deformable, allowing them to adapt their morphology in unstructured environments. This will require soft active materials for actuation, circuitry, and sensing of deformation and contact pressure. The emerging field of soft robotics utilizes these soft active materials to mimic the inherent compliance of natural soft-bodied systems. As the elasticity of robot components increases, the challenges for functionality revert to basic questions of fabrication, materials, and design - whereas such aspects are far more developed for traditional rigid-bodied systems. This thesis will highlight preliminary materials and designs that address the need for soft actuators and sensors, as well as emerging fabrication techniques for manufacturing stretchable circuits and devices based on liquid-embedded elastomers.
Earnest, James T; Hantak, Michael P; Park, Jung-Eun; Gallagher, Tom
2015-06-01
Coronaviruses (CoVs) and low-pathogenicity influenza A viruses (LP IAVs) depend on target cell proteases to cleave their viral glycoproteins and prime them for virus-cell membrane fusion. Several proteases cluster into tetraspanin-enriched microdomains (TEMs), suggesting that TEMs are preferred virus entry portals. Here we found that several CoV receptors and virus-priming proteases were indeed present in TEMs. Isolated TEMs, when mixed with CoV and LP IAV pseudoparticles, cleaved viral fusion proteins to fusion-primed fragments and potentiated viral transductions. That entering viruses utilize TEMs as a protease source was further confirmed using tetraspanin antibodies and tetraspanin short hairpin RNAs (shRNAs). Tetraspanin antibodies inhibited CoV and LP IAV infections, but their virus-blocking activities were overcome by expressing excess TEM-associated proteases. Similarly, cells with reduced levels of the tetraspanin CD9 resisted CoV pseudoparticle transductions but were made susceptible by overproducing TEM-associated proteases. These findings indicated that antibodies and CD9 depletions interfere with viral proteolytic priming in ways that are overcome by surplus proteases. TEMs appear to be exploited by some CoVs and LP IAVs for appropriate coengagement with cell receptors and proteases. Enveloped viruses use their surface glycoproteins to catalyze membrane fusion, an essential cell entry step. Host cell components prime these viral surface glycoproteins to catalyze membrane fusion at specific times and places during virus cell entry. Among these priming components are proteases, which cleave viral surface glycoproteins, unleashing them to refold in ways that catalyze virus-cell membrane fusions. For some enveloped viruses, these proteases are known to reside on target cell surfaces. This research focuses on coronavirus and influenza A virus cell entry and identifies TEMs as sites of viral proteolysis, thereby defining subcellular locations of virus
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.
Aloe vera in active and passive regions of electronic devices towards a sustainable development
Lim, Zhe Xi; Sreenivasan, Sasidharan; Wong, Yew Hoong; Cheong, Kuan Yew
2017-07-01
The increasing awareness towards sustainable development of electronics has driven the search for natural bio-organic materials in place of conventional electronic materials. The concept of using natural bio-organic materials in electronics provides not only an effective solution to address global electronic waste crisis, but also a compelling template for sustainable electronics manufacturing. This paper attempts to provide an overview of using Aloe vera gel as a natural bio-organic material for various electronic applications. Important concepts such as responses of living Aloe vera plant towards electrical stimuli and demonstrations of Aloe vera films as passive and active regions of electronic devices are highlighted in chronological order. The biodegradability and biocompatibility of Aloe vera can bring the world a step closer towards the ultimate goal of sustainable development of electronic devices from "all-natural" materials.
Gao, Gang; Xu, Xiao-Xia; Yu, Jing; Li, Lin-Miao; Ju, Wen-Yan; Jin, Feng-Liang; Freed, Shoaib
2016-09-01
The proteolytic activation of prophenoloxidase (proPO) is a humoral defense mechanism in insects and crustaceans. Phenoloxidase (PO) is produced as an inactive precursor namely, proPO and is activated via specific proteolytic cleavage by proPO-activating proteinase. The current research reports two novel serine proteinase genes (PxSP1-768 bp and PxSP2-816 bp) from Plutella xylostella, encoding 255 and 271 amino acid residues, respectively. Tissue distribution analyses by semiquantitative reverse transcription-PCR (RT-PCR) revealed the resultant genes to be primarily expressed in the hemocytes, while quantitative-RT-PCR (qRT-PCR) assay showed that transcription level of PxSP1 and PxSP2 increased significantly after injection of the fungal pathogen Beauveria bassiana. Purified recombinant fusion proteins of PxSP2 and PxSP1 were injected to New Zealand white rabbits and polyclonal antibodies were generated with the titers of 1:12,800. After silencing the expression of PxSP2 by RNAi, the PO activity decreased significantly. The results show that PxSP2 is involved in prophenoloxidase activation in P. xylostella. © 2016 Wiley Periodicals, Inc.
Leite Júnior, Bruno Ricardo de Castro; Tribst, Alline Artigiani Lima; Cristianini, Marcelo
2015-01-01
This study investigated the effect of high pressure homogenization (HPH) (up to 190 MPa) on porcine pepsin (proteolytic and milk-clotting activities), and the consequences of using the processed enzyme in milk coagulation and gel formation (rheological profile, proteolysis, syneresis, and microstructure). Although the proteolytic activity (PA) was not altered immediately after the HPH process, it reduced during enzyme storage, with a 5% decrease after 60 days of storage for samples obtained with the enzyme processed at 50, 100 and 150 MPa. HPH increased the milk-clotting activity (MCA) of the enzyme processed at 150 MPa, being 15% higher than the MCA of non-processed samples after 60 days of storage. The enzyme processed at 150 MPa produced faster aggregation and a more consistent milk gel (G’ value 92% higher after 90 minutes) when compared with the non-processed enzyme. In addition, the gels produced with the enzyme processed at 150 MPa showed greater syneresis after 40 minutes of coagulation (forming a more compact protein network) and lower porosity (evidenced by confocal microscopy). These effects on the milk gel can be associated with the increment in MCA and reduction in PA caused by the effects of HPH on pepsin during storage. According to the results, HPH stands out as a process capable of changing the proteolytic characteristics of porcine pepsin, with improvements on the milk coagulation step and gel characteristics. Therefore, the porcine pepsin submitted to HPH process can be a suitable alternative for the production of cheese. PMID:25938823
Active pixel sensor array as a detector for electron microscopy.
Milazzo, Anna-Clare; Leblanc, Philippe; Duttweiler, Fred; Jin, Liang; Bouwer, James C; Peltier, Steve; Ellisman, Mark; Bieser, Fred; Matis, Howard S; Wieman, Howard; Denes, Peter; Kleinfelder, Stuart; Xuong, Nguyen-Huu
2005-09-01
A new high-resolution recording device for transmission electron microscopy (TEM) is urgently needed. Neither film nor CCD cameras are systems that allow for efficient 3-D high-resolution particle reconstruction. We tested an active pixel sensor (APS) array as a replacement device at 200, 300, and 400 keV using a JEOL JEM-2000 FX II and a JEM-4000 EX electron microscope. For this experiment, we used an APS prototype with an area of 64 x 64 pixels of 20 microm x 20 microm pixel pitch. Single-electron events were measured by using very low beam intensity. The histogram of the incident electron energy deposited in the sensor shows a Landau distribution at low energies, as well as unexpected events at higher absorbed energies. After careful study, we concluded that backscattering in the silicon substrate and re-entering the sensitive epitaxial layer a second time with much lower speed caused the unexpected events. Exhaustive simulation experiments confirmed the existence of these back-scattered electrons. For the APS to be usable, the back-scattered electron events must be eliminated, perhaps by thinning the substrate to less than 30 microm. By using experimental data taken with an APS chip with a standard silicon substrate (300 microm) and adjusting the results to take into account the effect of a thinned silicon substrate (30 microm), we found an estimate of the signal-to-noise ratio for a back-thinned detector in the energy range of 200-400 keV was about 10:1 and an estimate for the spatial resolution was about 10 microm.
Relationship between electronic structure and radioprotective activity of some indazoles
International Nuclear Information System (INIS)
Sokolov, Yu.A.
2000-01-01
The quantum-chemical study of electronic structure of 29 indasoles with complete optimization of geometry and search of quantitative link between the established characteristics and radioprotective activity (RPA) was carried out through the MNDO method with application of multiple linear and nonlinear regression analysis and the basic component method. The equations of correlation relationship between the RPA and electronic characteristics are presented. 10 indasole structures, the forecasted RPA values whereof (survival rate, %) equal 50% and above, are selected. The statistic significance of the obtained correlation equations and their regression coefficients make it possible to conclude, that the established relationships are not accidental and are prospective for forecasting RPA of other close compounds of the indasole series [ru
Electronic states of carbon alloy catalysts and nitrogen substituent effects on catalytic activity
Hata, Tomoyuki; Ushiyama, Hiroshi; Yamashita, Koichi
2013-03-01
In recent years, Carbon Alloy Catalysts (CACs) are attracting attention as a candidate for non-platinum-based cathode catalysts in fuel cells. Oxygen reduction reactions at the cathode are divided into two elementary processes, electron transfer and oxygen adsorption. The electron transfer reaction is the rate-determining, and by comparison of energy levels, catalytic activity can be evaluated quantitatively. On the other hand, to begin with, adsorption mechanism is obscure. The purpose of this study is to understand the effect of nitrogen substitution and oxygen adsorption mechanism, by first-principle electronic structure calculations for nitrogen substituted models. To reproduce the elementary processes of oxygen adsorption, we assumed that the initial structures are formed based on the Pauling model, a CACs model and nitrogen substituted CACs models in which various points are replaced with nitrogen. When we try to focus only on the DOS peaks of oxygen, in some substituted model that has high adsorption activity, a characteristic partial occupancy state was found. We conclude that this state will affect the adsorption activity, and discuss on why partially occupied states appear with simplification by using an orbital correlation diagram.
Directory of Open Access Journals (Sweden)
Malinowska-Pańczyk Edyta
2018-06-01
Full Text Available The aim of this study was to determine changes in the activity of proteolytic enzymes and transglutaminase of fish and mammal meat after pressurization at subzero temperatures. The activity was measured at the optimal pHs determined for enzymes from particular types of tested meat. It was found that increasing the pressure in the range of 60-193 MPa, did not change significantly the activity of acidic proteases of cod flesh, while the activity of neutral and alkaline proteases decreased drastically. Proteolytic enzymes from salmon flesh were more resistant than those from cod flesh. They maintained or increased (neutral protease activity after pressurization. The activity of the endogenous enzymes of bovine meat increased with pressure increase, except for acidic proteases, the activity of which was reduced after treatment at 193 MPa to the level similar to unpressurized meat. Endogenous proteases of porcine meat were activated by high-pressure treatment. It has been shown that activity of TGase in unpressurized flesh from cod was 5 times higher than that from unpressurized salmon. Depending on the type of meat, these enzymes were also significantly different in their sensitivity to pressure. The pressure of 60 and 193 MPa led to a complete inactivation of the TGase in cod flesh, while the activity of salmon flesh TGase was decreased only by 15 and 21%, respectively.
Brzęk, Anna; Strauss, Markus; Przybylek, Bianca; Dworrak, Tarja; Dworrak, Birgit; Leischik, Roman
2018-01-01
Apart from many positive changes associated with technical civilization, there are also - from the health point of view - some threats. The reduction in the level of physical activity is one of them. The aim of the study was to investigate whether there are any relationships between children's physical activity and behaviors, and to assess the impact of the adults' activity on their children's habits. The study involved a group of 340 children aged 7-12 years (mean age: 9.81 ±1.7) and their parents. In order to evaluate children's physical activity and the amount of time that they spend with electronic devices, an original questionnaire and the IPAQ questionnaire were used. Children usually use electronic devices between 2 and 7 days per week (mean: 4.74 ±0.86), regardless of sex ( p > 0.09) and spend between 5 and 1620 min per week (mean: 459.46 ±308.1) with their mobile phone, tablet, PCs and TVs. 67.92% of boys and 69.61% of girls lead an active lifestyle. The children's activity level depends on their parents' level of activity ( p Parents of semi-active children lead a lifestyle with a moderate level of physical activity. The level of physical activity in younger children depends on the children's relationship with their parents and their level of activity. Children spend a lot of free time with their electronic devices. It is necessary to develop and implement activities intended to raise awareness of children and their families about the effects of hypokinesis.
Active electronic personal dosemeter in interventional radiology
International Nuclear Information System (INIS)
Prlic, I.; Suric Mihic, M.; Vucic, Z.
2008-01-01
A recently developed active electronic personal dosemeter (AEPD) was utilised in order to measure the levels and the structure of occupational exposure to scattered X-ray radiation of medical staff who performed percutaneous revascularisation therapy that involves interventional radiology (IR) on the pelvis and upper leg arteries. The AEPDs, placed on the operators' and assistants' chests, that is, above the protective apron, continuously measured and recorded the received doses and, as a novelty, dose rates as a function of time, thus yielding a unique record of occupational doses and dose rates pattern at the working place. This paper presents and discusses one typical daily pattern in which seven percutaneous interventions were performed. (authors)
Energy Technology Data Exchange (ETDEWEB)
Sapnar, K.B.; Dhole, S.D. [Department of Physics, University of Pune, Pune 411007 (India); Bhoraskar, V.N., E-mail: vnb@physics.unipune.ac.in [Department of Physics, University of Pune, Pune 411007 (India)
2012-04-01
Highlights: Black-Right-Pointing-Pointer The phases of TiO{sub 2} nanoparticles have been changed by electron irradiation. Black-Right-Pointing-Pointer The photocatalytic activity of TiO{sub 2} gets enhanced after electron irradiation. Black-Right-Pointing-Pointer The brookite phase has appeared in TiO{sub 2} after electron irradiation. - Abstract: Samples of TiO{sub 2} nanoparticles, with mixed anatase and rutile phases, were irradiated with 6.5 MeV electrons at fluences, 1.5, 2.0, 2.5, 3.0, and 3.5 Multiplication-Sign 10{sup 15} e cm{sup -2} and characterized by several methods. With increasing electron fluence, a continuous decrease in the average particle size from {approx}80 nm to around 30 nm were observed along with a decrease in the rutile and the anatase phases of TiO{sub 2}, but at different rates, and growth of the TiO{sub 2} brookite phase at slow rate. The photocatalytic activities of different electron irradiated TiO{sub 2} samples, in the photodegradation of methylene blue, were studied by recording UV-Vis absorption spectra of the respective solutions. On electron irradiation, even though the rutile phase in the TiO{sub 2} was decreasing, the photocatalytic activity of the nanoparticles increased continuously with fluence up to {approx}3.0 Multiplication-Sign 10{sup 15} e cm{sup -2}, but decreased at 3.5 Multiplication-Sign 10{sup 15} e cm{sup -2}. The energy levels introduced by the brookite phase and the electron induced defects in TiO{sub 2} could have effectively reduced the electron-hole recombination rate in the absence of the rutile phase. The observed enhancement in the photocatalytic activity of the irradiated TiO{sub 2} is attributed to the formation of small size particles, the introduction of the oxygen related vacancies and other defects, the growth of the brookite phase, and increased absorption of radiation over the ultraviolet and visible range.
Dimoglo, A S; Chumakov, Y M; Dobrova, B N; Saracoglu, M
1997-04-01
In the frameworks of the electron-topological method (ETM) the structure-antitumor activity relationship was investigated for a series of thiosemicarbazone derivatives. The series included 70 compounds. Conformational analysis and quantum-chemical calculations were carried out for each compound. The revealed activity feature showed a satisfactory description of the class of active compounds according to two different parameters P and alpha estimating the probabilities of the feature realization in the class of active compounds (they are equal to 0.94 and 0.86, correspondingly). The results of testing demonstrated the high ability of ETM in predicting the activity investigated.
Effect of Electronic Messaging on Physical Activity Participation among Older Adults
Antoine Parker, Chantrell; Ellis, Rebecca
2016-01-01
The purpose of this study was to determine if electronic messaging would increase min of aerobic physical activity (PA) among older adults. Participants were active older adults (n = 28; M age = 60 years, SD = 5.99, and range = 51?74 years). Using an incomplete within-subjects crossover design, participants were randomly assigned to begin the 4-week study receiving the treatment condition (a morning and evening text message) or the control condition (an evening text message). Participants sel...
Directory of Open Access Journals (Sweden)
Suntinanalert, P.
2007-03-01
Full Text Available Five isolates of bacteria with high proteolytic activity, isolated from water samples of intensive shrimp ponds in southern Thailand, were selected to test for the ability to control the shrimp pathogen Vibrioharveyi. 70 μl of each culture broth were investigated for their ability to inhibit V. harveyi using an agar well diffusion test but only one isolate W3 gave a reasonable sized inhibition zone of 21.62 mm. This zone wassimilar to that of oxolinic acid (2 μg and sulfamethoxazole (25 μg. The W3 isolate was identified as Pseudomonas sp. Shrimp cultivation in aquaria was conducted to investigate the inhibition of V. harveyi bythe isolate W3. The experiment consisted of a treatment of the shrimp culture with an inoculum of the isolate W3 and V. harveyi (biocontrol set, a positive control set (only inoculation of V. harveyi and a negativecontrol set as without inoculation. No mortality was found in the negative control. Shrimp mortality in the biocontrol set (33% was lower than that in the positive control set (40%; however, it showed no significantdifference (p>0.05. The average numbers of V. harveyi over 12 days of the biocontrol set were lower than those in the positive control set by about 1 log cycle although the numbers were not significantly different(p>0.05. The shrimp growth rate at day 32 of cultivation was in order of the biocontrol treatment (10.17% > the negative control treatment (9.44% > the positive control set (9.28%, but no significant difference (p>0.05 was observed among treatments.
Interdomain electron transfer in cellobiose dehydrogenase is governed by surface electrostatics.
Kadek, Alan; Kavan, Daniel; Marcoux, Julien; Stojko, Johann; Felice, Alfons K G; Cianférani, Sarah; Ludwig, Roland; Halada, Petr; Man, Petr
2017-02-01
Cellobiose dehydrogenase (CDH) is a fungal extracellular oxidoreductase which fuels lytic polysaccharide monooxygenase with electrons during cellulose degradation. Interdomain electron transfer between the flavin and cytochrome domain in CDH, preceding the electron flow to lytic polysaccharide monooxygenase, is known to be pH dependent, but the exact mechanism of this regulation has not been experimentally proven so far. To investigate the structural aspects underlying the domain interaction in CDH, hydrogen/deuterium exchange (HDX-MS) with improved proteolytic setup (combination of nepenthesin-1 with rhizopuspepsin), native mass spectrometry with ion mobility and electrostatics calculations were used. HDX-MS revealed pH-dependent changes in solvent accessibility and hydrogen bonding at the interdomain interface. Electrostatics calculations identified these differences to result from charge neutralization by protonation and together with ion mobility pointed at higher electrostatic repulsion between CDH domains at neutral pH. In addition, we uncovered extensive O-glycosylation in the linker region and identified the long-unknown exact cleavage point in papain-mediated domain separation. Transition of CDH between its inactive (open) and interdomain electron transfer-capable (closed) state is shown to be governed by changes in the protein surface electrostatics at the domain interface. Our study confirms that the interdomain electrostatic repulsion is the key factor modulating the functioning of CDH. The results presented in this paper provide experimental evidence for the role of charge repulsion in the interdomain electron transfer in cellobiose dehydrogenases, which is relevant for exploiting their biotechnological potential in biosensors and biofuel cells. Copyright © 2016 Elsevier B.V. All rights reserved.
Effect of Electronic Messaging on Physical Activity Participation among Older Adults
Directory of Open Access Journals (Sweden)
Chantrell Antoine Parker
2016-01-01
Full Text Available The purpose of this study was to determine if electronic messaging would increase min of aerobic physical activity (PA among older adults. Participants were active older adults (n=28; M age = 60 years, SD = 5.99, and range = 51–74 years. Using an incomplete within-subjects crossover design, participants were randomly assigned to begin the 4-week study receiving the treatment condition (a morning and evening text message or the control condition (an evening text message. Participants self-reported min of completed aerobic PA by cell phone text. The 1-way within-subjects ANOVA showed significant group differences (p<0.05. Specifically, when participants were in the treatment condition, they reported significantly greater average weekly min of aerobic PA (M = 96.88 min, SD = 62.9 compared to when they completed the control condition (M = 71.68 min, SD = 40.98. Electronic messaging delivered via cell phones was effective at increasing min of aerobic PA among older adults.
Findeisen, Peter; Thumfart, Jörg Oliver; Costina, Victor; Hofheinz, Ralf; Neumaier, Michael
2013-09-01
To determine the preanalytical quality of serum and plasma by monitoring the time-dependent ex vivo decay of a synthetic reporter peptide (RP) with liquid chromatography/mass spectrometry (LC/MS). Serum and plasma specimens were spiked with the RP and proteolytic fragments were monitored with LC/MS at different preanalytical time points ranging from 2 to 24 hours after blood withdrawal. The concentration of fragments changed in a time-dependent manner, and respective peptide profiles were used to classify specimens according to their preanalytical time span. Classification accuracy was high, with values always above 0.89 for areas under receiver operating characteristic curves. This "proteomics degradation clock" can be used to estimate the preanalytical quality of serum and plasma and might have impact on quality control procedures of biobanking repositories.
Solli, Ann Iren; Fadnes, Bodil; Winberg, Jan-Olof; Uhlin-Hansen, Lars
2013-01-01
Matrix metalloproteinase 2 (MMP-2) is a proteolytic enzyme that degrades extracellular matrix proteins. Recent studies indicate that MMP-2 also has a role in intracellular proteolysis during various pathological conditions, such as ischemic injuries in heart and brain and in tumor growth. The present study was performed to map the distribution of intracellular MMP-2 activity in various mouse tissues and cells under physiological conditions. Samples from normal brain, heart, lung, liver, spleen, pancreas, kidney, adrenal gland, thyroid gland, gonads, oral mucosa, salivary glands, esophagus, intestines, and skin were subjected to high-resolution in situ gelatin zymography and immunohistochemical staining. In hepatocytes, cardiac myocytes, kidney tubuli cells, epithelial cells in the oral mucosa as well as in excretory ducts of salivary glands, and adrenal cortical cells, we found strong intracellular gelatinolytic activity that was significantly reduced by the metalloprotease inhibitor EDTA but not by the cysteine protease inhibitor E-64. Furthermore, the gelatinolytic activity was co-localized with MMP-2. Western blotting and electron microscopy combined with immunogold labeling revealed the presence of MMP-2 in different intracellular compartments of isolated hepatocytes. Our results indicate that MMP-2 takes part in intracellular proteolysis in specific tissues and cells during physiological conditions. PMID:23482328
Baker, L. Robert
2011-08-18
The role of the oxide-metal interface in determining the activity and selectivity of chemical reactions catalyzed by metal particles on an oxide support is an important topic in science and industry. A proposed mechanism for this strong metal-support interaction is electronic activation of surface adsorbates by charge carriers. Motivated by the goal of using electronic activation to drive nonthermal chemistry, we investigated the ability of the oxide support to mediate charge transfer. We report an approximately 2-fold increase in the turnover rate of catalytic carbon monoxide oxidation on platinum nanoparticles supported on stoichiometric titanium dioxide (TiO2) when the TiO2 is made highly n-type by fluorine (F) doping. However, for nonstoichiometric titanium oxide (TiOX<2) the effect of F on the turnover rate is negligible. Studies of the titanium oxide electronic structure show that the energy of free electrons in the oxide determines the rate of reaction. These results suggest that highly n-type TiO2 electronically activates adsorbed oxygen (O) by electron spillover to form an active O- intermediate. © 2011 American Chemical Society.
Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael
2013-01-01
ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley
Hoyte, Ashley C; Jamin, Augusta V; Koneru, Pratibha C; Kobe, Matthew J; Larue, Ross C; Fuchs, James R; Engelman, Alan N; Kvaratskhelia, Mamuka
2017-12-01
The pyridine-based multimerization selective HIV-1 integrase (IN) inhibitors (MINIs) are a distinct subclass of allosteric IN inhibitors. MINIs potently inhibit HIV-1 replication during virion maturation by inducing hyper- or aberrant IN multimerization but are largely ineffective during the early steps of viral replication. Here, we investigated the mechanism for the evolution of a triple IN substitution (T124N/V165I/T174I) that emerges in cell culture with a representative MINI, KF116. We show that HIV-1 NL4-3(IN T124N/V165I/T174I) confers marked (>2000-fold) resistance to KF116. Two IN substitutions (T124N/T174I) directly weaken inhibitor binding at the dimer interface of the catalytic core domain but at the same time markedly impair HIV-1 replication capacity. Unexpectedly, T124N/T174I IN substitutions inhibited proteolytic processing of HIV-1 polyproteins Gag and Gag-Pol, resulting in immature virions. Strikingly, the addition of the third IN substitution (V165I) restored polyprotein processing, virus particle maturation, and significant levels of replication capacity. These results reveal an unanticipated role of IN for polyprotein proteolytic processing during virion morphogenesis. The complex evolutionary pathway for the emergence of resistant viruses, which includes the need for the compensatory V165I IN substitution, highlights a relatively high genetic barrier exerted by MINI KF116. Additionally, we have solved the X-ray structure of the drug-resistant catalytic core domain protein, which provides means for rational development of second-generation MINIs. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.
DEFF Research Database (Denmark)
Wang, Bijue; Chen, Pingping; Jensen, Anne-Christine Bay
2009-01-01
BACKGROUND: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP- and aggreca......BACKGROUND: Progressive loss of articular cartilage is a central hallmark in many joint disease, however, the relative importance of individual proteolytic pathways leading to cartilage erosion is at present unknown. We therefore investigated the time-dependant release ex vivo of MMP......- and aggrecanase-derived fragments of aggrecan and type II collagen into the supernatant of bovine cartilage explants cultures using neo-epitope specific immunoassays, and to associate the release of these fragments with the activity of proteolytic enzymes using inhibitors. FINDINGS: Bovine cartilage explants were...... cultured in the presence or absence of the catabolic cytokines oncostatin M (OSM) and tumor necrosis factor alpha (TNFalpha). In parallel, explants were co-cultured with protease inhibitors such as GM6001, TIMP1, TIMP2 and TIMP3. Fragments released into the supernatant were determined using a range of neo...
Ponomarenko, Natalia A; Durova, Oxana M; Vorobiev, Ivan I; Belogurov, Alexey A; Telegin, Georgy B; Suchkov, Sergey V; Misikov, Victor K; Morse, Herbert C; Gabibov, Alexander G
2006-02-28
Autoantibodies toward myelin basic protein (MBP) evidently emerge in sera and cerebrospinal fluid of the patients with multiple sclerosis (MS), as well as in a MS rodent model, i.e., experimental autoimmune encephalomyelitis (EAE). The studies of the last two decades have unveiled somewhat controversial data on the diagnostic applicability of anti-MBP autoantibodies as a disease' marker. Here, we present the results of new functional analysis of the anti-MBP autoantibodies isolated from MS (in patients) and EAE (in mice) sera, based on their proteolytic activity against the targeted autoantigen. The activity was shown to be the intrinsic property of the IgG molecule. No activity was found in the sera-derived antibody fraction of healthy donors and control mice. Sera of 24 patients with clinically proven MS at different stages of the disease, and 20 healthy controls were screened for the anti-MBP antibody-mediated proteolytic activity. The activity correlated with the scores on the MS expanded disability status scale (EDSS) (r(2)=0.85, P<0.001). Thus, the anti-MBP autoantibody-mediated proteolysis may be regarded as an additional marker of the disease progression.
Autoproteolytic Activation of Bacterial Toxins
Directory of Open Access Journals (Sweden)
Aimee Shen
2010-05-01
Full Text Available Protease domains within toxins typically act as the primary effector domain within target cells. By contrast, the primary function of the cysteine protease domain (CPD in Multifunctional Autoprocessing RTX-like (MARTX and Clostridium sp. glucosylating toxin families is to proteolytically cleave the toxin and release its cognate effector domains. The CPD becomes activated upon binding to the eukaryotic-specific small molecule, inositol hexakisphosphate (InsP6, which is found abundantly in the eukaryotic cytosol. This property allows the CPD to spatially and temporally regulate toxin activation, making it a prime candidate for developing anti-toxin therapeutics. In this review, we summarize recent findings related to defining the regulation of toxin function by the CPD and the development of inhibitors to prevent CPD-mediated activation of bacterial toxins.
Energy Technology Data Exchange (ETDEWEB)
Mueller, H M; Edlich, W; Lyr, H
1986-01-01
As a model system for the investigation of peroxidative effects in the phytopathogenic fungi Botrytis cinerea in submers culture was used. The influence was investigated of hydrogenperoxide with biochemical and cytological methods. The hyphae showed significant changes of the ultrastructure: swelling and vacuolization of mitochondria, loss of ribosomes, vesiculation of endoplasmic reticulum, degradation and lysis of the cell ribosomes, and deposition of lipid droplets on the membranes and in the cytoplasm, dependent on the concentration of the peroxide. Same effects with higher injuries of the membrane system are found in Phytophthorea infestans. A parallel investigation on fibroblasts showed similar effects: pathological changes of mitochondria, but also pycnosis in the nucleus. Comparative ultrastructure investigations were performed in cultures of Phytophthora infestans with tetrachlorcarbon and chloroneb, a specific fungicide substance with a new mode of action. Scavengers like ..cap alpha..-tocopherol and piperonyl butoxide inhibit the toxic effect of the investigated substances in various degrees. Results are discussed in regard to the effect of a lipid peroxidative and proteolytic attack in pesticides of unknown mechanism of action.
Does plasmin have anticoagulant activity?
Directory of Open Access Journals (Sweden)
Jane Hoover-Plow
2010-03-01
Full Text Available Jane Hoover-PlowJoseph J Jacobs Center for Thrombosis and Vascular Biology, Departments of Cardiovascular Medicine and Molecular Cardiology, Lerner Research Institute Cleveland Clinic, Ohio, USAAbstract: The coagulation and fibrinolytic pathways regulate hemostasis and thrombosis, and an imbalance in these pathways may result in pathologic hemophilia or thrombosis. The plasminogen system is the primary proteolytic pathway for fibrinolysis, but also has important proteolytic functions in cell migration, extracellular matrix degradation, metalloproteinase activation, and hormone processing. Several studies have demonstrated plasmin cleavage and inactivation of several coagulation factors, suggesting plasmin may be not only be the primary fibrinolytic enzyme, but may have anticoagulant properties as well. The objective of this review is to examine both in vitro and in vivo evidence for plasmin inactivation of coagulation, and to consider whether plasmin may act as a physiological regulator of coagulation. While several studies have demonstrated strong evidence for plasmin cleavage and inactivation of coagulation factors FV, FVIII, FIX, and FX in vitro, in vivo evidence is lacking for a physiologic role for plasmin as an anticoagulant. However, inactivation of coagulation factors by plasmin may be useful as a localized anticoagulant therapy or as a combined thrombolytic and anticoagulant therapy.Keywords: thrombosis, anticoagulant, cardiovascular disease, plasminogen’s protease, blood
Yun, Eun-Tae; Yoo, Ha-Young; Bae, Hyokwan; Kim, Hyoung-Il; Lee, Jaesang
2017-09-05
This study elucidates the mechanism behind persulfate activation by exploring the role of various oxyanions (e.g., peroxymonosulfate, periodate, and peracetate) in two activation systems utilizing iron nanoparticle (nFe 0 ) as the reducing agent and single-wall carbon nanotubes (CNTs) as electron transfer mediators. Since the tested oxyanions serve as both electron acceptors and radical precursors in most cases, oxidative degradation of organics was achievable through one-electron reduction of oxyanions on nFe 0 (leading to radical-induced oxidation) and electron transfer mediation from organics to oxyanions on CNTs (leading to oxidative decomposition involving no radical formation). A distinction between degradative reaction mechanisms of the nFe 0 /oxyanion and CNT/oxyanion systems was made in terms of the oxyanion consumption efficacy, radical scavenging effect, and EPR spectral analysis. Statistical study of substrate-specificity and product distribution implied that the reaction route induced on nFe 0 varies depending on the oxyanion (i.e., oxyanion-derived radical), whereas the similar reaction pathway initiates organic oxidation in the CNT/oxyanion system irrespective of the oxyanion type. Chronoamperometric measurements further confirmed electron transfer from organics to oxyanions in the presence of CNTs, which was not observed when applying nFe 0 instead.
CdWO4 polymorphs: Selective preparation, electronic structures, and photocatalytic activities
International Nuclear Information System (INIS)
Yan, Tingjiang; Li, Liping; Tong, Wenming; Zheng, Jing; Wang, Yunjian; Li, Guangshe
2011-01-01
This work explored the selective synthesis of polymorphs of CdWO 4 in either tetragonal or monoclinic phase by optimizing the experimental parameters. Systematic characterization indicated that both polymorphs possessed similar spherical morphologies but different structural building blocks. Electronic structures calculations for both polymorphs demonstrated the same constructions of conduction band or valence band, while the conduction band widths of both polymorphs were quite different. Both CdWO 4 polymorphs exhibited good photocatalytic activity for degradation of methyl orange under UV light irradiation. When comparing to some other well-known tungstate oxide materials, the photocatalytic activity was found to follow such a consequence, monoclinic CdWO 4 ∼monoclinic ZnWO 4 >tetragonal CdWO 4 >tetragonal CaWO 4 . The specific photocatalytic activity of monoclinic CdWO 4 was even higher than that of commercial TiO 2 photocatalyst (Degussa P25). The increased activity from the tetragonal CdWO 4 to the monoclinic was consistent with the trend of the decreased symmetry, and this could be explained in terms of the geometric structures and electronic structures for both polymorphs. -- Graphical abstract: Monoclinic CdWO 4 exhibited a much higher photocatalytic activity than the tetragonal form owing to the lower symmetry, more distorted geometric structure, and the dispersive band configuration. Display Omitted Research highlights: → Polymorphs of CdWO 4 in either tetragonal or monoclinic phase were selectively synthesized. → Both polymorphs possessed similar spherical morphologies, while the relevant structural building blocks were different. → Photocatalytic activities of CdWO 4 polymorphs depended strongly on the symmetry, geometric structure, as well as band configuration.
Hsu, Liang-Yan; Wu, Ning; Rabitz, Herschel
2016-11-30
We investigate electron transport through series and parallel intramolecular circuits in the framework of the multi-level Redfield theory. Based on the assumption of weak monomer-bath couplings, the simulations depict the length and temperature dependence in six types of intramolecular circuits. In the tunneling regime, we find that the intramolecular circuit rule is only valid in the weak monomer coupling limit. In the thermally activated hopping regime, for circuits based on two different molecular units M a and M b with distinct activation energies E act,a > E act,b , the activation energies of M a and M b in series are nearly the same as E act,a while those in parallel are nearly the same as E act,b . This study gives a comprehensive description of electron transport through intramolecular circuits from tunneling to thermally activated hopping. We hope that this work can motivate additional studies to design intramolecular circuits based on different types of building blocks, and to explore the corresponding circuit laws and the length and temperature dependence of conductance.
DEFF Research Database (Denmark)
Miyagi, Haruhide; Madsen, Lars Bojer
2013-01-01
We present the time-dependent restricted-active-space self-consistent-field (TD-RASSCF) theory as a framework for the time-dependent many-electron problem. The theory generalizes the multiconfigurational time-dependent Hartree-Fock (MCTDHF) theory by incorporating the restricted-active-space scheme...... well known in time-independent quantum chemistry. Optimization of the orbitals as well as the expansion coefficients at each time step makes it possible to construct the wave function accurately while using only a relatively small number of electronic configurations. In numerical calculations of high...
12 CFR 7.5003 - Composite authority to engage in electronic activities.
2010-01-01
... 12 Banks and Banking 1 2010-01-01 2010-01-01 false Composite authority to engage in electronic activities. 7.5003 Section 7.5003 Banks and Banking COMPTROLLER OF THE CURRENCY, DEPARTMENT OF THE TREASURY... itself part of or incidental to the business of banking or is otherwise permissible under Federal law. ...
DEFF Research Database (Denmark)
Lund, Ida K; Jögi, Annika; Rønø, Birgitte
2008-01-01
highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only...
DEFF Research Database (Denmark)
Peters, Diane E; Szabo, Roman; Friis, Stine
2014-01-01
The membrane-anchored serine protease prostasin (CAP1/PRSS8) is part of a cell surface proteolytic cascade that is essential for epithelial barrier formation and homeostasis. Here, we report the surprising finding that prostasin executes these functions independent of its own enzymatic activity. ...
Ferrari, Gerson Luis de Moraes; Araújo, Timóteo Leandro; Oliveira, Luis Carlos; Matsudo, Victor; Fisberg, Mauro
2015-01-01
To describe the association between electronic devices in the bedroom with sedentary time and physical activity, both assessed by accelerometry, in addition to body mass index in children from São Caetano do Sul. The sample consisted of 441 children. The presence of electronic equipment (television, personal computer, and videogames) in the bedroom was assessed by a questionnaire. For seven consecutive days, children used an accelerometer to objectively monitor the sedentary time and moderate-to-vigorous physical activity. Body mass index was categorized as suggested by the World Health Organization. Overall, 73.9%, 54.2% and 42.8% of children had TV, computer, and videogames in the bedroom, respectively, and spent an average of 500.7 and 59.1 min/day of sedentary time and moderate-to-vigorous physical activity. Of the children, 45.3% were overweight/obese. Girls with a computer in the bedroom (45 min/day) performed less moderate-to-vigorous physical activity than those without it (51.4 min/day). Similar results were observed for body mass index in boys. Moderate-to-vigorous physical activity was higher and body mass index was lower in children that had no electronic equipment in the bedroom. Presence of a computer (β=-4.798) and the combination TV+computer (β=-3.233) were negatively associated with moderate-to-vigorous physical activity. Videogames and the combinations with two or three electronic devices were positively associated with body mass index. Sedentary time was not associated with electronic equipment. Electronic equipment in the children's bedroom can negatively affect moderate-to-vigorous physical activity and body mass index regardless of gender, school, and annual family income, which can contribute to physical inactivity and childhood obesity. Copyright © 2015 Sociedade Brasileira de Pediatria. Published by Elsevier Editora Ltda. All rights reserved.
Koopmans, Timo; Wood, Thomas M.; 't Hart, Peter; Kleijn, Laurens H. J.; Hendrickx, Antoni P. A.; Willems, Rob J. L.; Breukink, Eefjan; Martin, Nathaniel I.
2015-01-01
The lipid II-binding N-terminus of nisin, comprising the so-called A/B ring system, was synthetically modified to provide antibacterially active and proteolytically stable derivatives. A variety of lipids were coupled to the C-terminus of the nisin A/B ring system to generate semisynthetic
Baker, L. Robert; Hervier, Antoine; Seo, Hyungtak; Kennedy, Griffin; Komvopoulos, Kyriakos; Somorjai, Gabor A.
2011-01-01
-support interaction is electronic activation of surface adsorbates by charge carriers. Motivated by the goal of using electronic activation to drive nonthermal chemistry, we investigated the ability of the oxide support to mediate charge transfer. We report
Iwata, Yuji; Ashida, Makoto; Hasegawa, Chisa; Tabara, Kazuki; Mishiba, Kei-Ichiro; Koizumi, Nozomu
2017-08-01
The unfolded protein response (UPR) is a homeostatic cellular response conserved in eukaryotic cells to alleviate the accumulation of unfolded proteins in the endoplasmic reticulum (ER). Arabidopsis bZIP28 is a membrane-bound transcription factor activated by proteolytic cleavage in response to ER stress, thereby releasing its cytosolic portion containing the bZIP domain from the membrane to translocate into the nucleus where it induces the transcription of genes encoding ER-resident molecular chaperones and folding enzymes. It has been widely recognized that the proteolytic activation of bZIP28 is mediated by the sequential cleavage of site-1 protease (S1P) and site-2 protease (S2P). In the present study we provide evidence that bZIP28 protein is cleaved by S2P, but not by S1P. We demonstrated that wild-type and s1p mutant plants produce the active, nuclear form of bZIP28 in response to the ER stress inducer tunicamycin. In contrast, tunicamycin-treated s2p mutants do not accumulate the active, nuclear form of bZIP28. Consistent with these observations, s2p mutants, but not s1p mutants, exhibited a defective transcriptional response of ER stress-responsive genes and significantly higher sensitivity to tunicamycin. Interestingly, s2p mutants accumulate two membrane-bound bZIP28 fragments with a shorter ER lumen-facing C-terminal domain. Importantly, the predicted cleavage sites are located far from the canonical S1P recognition motif previously described. We propose that ER stress-induced proteolytic activation of bZIP28 is mediated by the sequential actions of as-yet-unidentified protease(s) and S2P, and does not require S1P. © 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.
Automatic prediction of rheumatoid arthritis disease activity from the electronic medical records.
Directory of Open Access Journals (Sweden)
Chen Lin
Full Text Available We aimed to mine the data in the Electronic Medical Record to automatically discover patients' Rheumatoid Arthritis disease activity at discrete rheumatology clinic visits. We cast the problem as a document classification task where the feature space includes concepts from the clinical narrative and lab values as stored in the Electronic Medical Record.The Training Set consisted of 2792 clinical notes and associated lab values. Test Set 1 included 1749 clinical notes and associated lab values. Test Set 2 included 344 clinical notes for which there were no associated lab values. The Apache clinical Text Analysis and Knowledge Extraction System was used to analyze the text and transform it into informative features to be combined with relevant lab values.Experiments over a range of machine learning algorithms and features were conducted. The best performing combination was linear kernel Support Vector Machines with Unified Medical Language System Concept Unique Identifier features with feature selection and lab values. The Area Under the Receiver Operating Characteristic Curve (AUC is 0.831 (σ = 0.0317, statistically significant as compared to two baselines (AUC = 0.758, σ = 0.0291. Algorithms demonstrated superior performance on cases clinically defined as extreme categories of disease activity (Remission and High compared to those defined as intermediate categories (Moderate and Low and included laboratory data on inflammatory markers.Automatic Rheumatoid Arthritis disease activity discovery from Electronic Medical Record data is a learnable task approximating human performance. As a result, this approach might have several research applications, such as the identification of patients for genome-wide pharmacogenetic studies that require large sample sizes with precise definitions of disease activity and response to therapies.
Energy Technology Data Exchange (ETDEWEB)
Kim, Jong-Wan [Department of Food Science and Biotechnology, Kyungnam University, Masan 631-701 (Korea, Republic of); Lee, Byung Cheol [Laboratory for Quantum Optics, Korea Atomic Energy Research Institute, Daejeon 305-600 (Korea, Republic of); Lee, Jong-Hwa [School of Bioresource Sciences, Andong National University, Andong 760-749 (Korea, Republic of); Nam, Ki-Chang [Chemistry and Biotechnology Examinations Bureau, Korean Intellectual Property Office, Daejeon 302-701 (Korea, Republic of); Lee, Seung-Cheol [Department of Food Science and Biotechnology, Kyungnam University, Masan 631-701 (Korea, Republic of)], E-mail: sclee@kyungnam.ac.kr
2008-01-15
After electron-beam irradiation of citrus pomaces (CP), the total phenolic content (TPC), radical scavenging activity (RSA), and reducing power (RP) were evaluated. When CP were irradiated at 37.9 kGy; the TPC, RSA and RP of water extract of CP increased from 6543.2 to 7405.4 {mu}M, 37.6% to 52.9%, and 0.64 to 0.90, respectively, compared with the non-irradiated control. The results indicate that the electron-beam irradiation can be an efficient process for increasing the antioxidant activity of CP.
Electronics department progress report. Basic and applied research activity 1975-1977
International Nuclear Information System (INIS)
1978-10-01
The electronics department at Risoe National Laboratory, Denmark, comprises about 80 people and is made up of essentially two main divisions. One deals with the provision of instrumentation and measurement expertise for the different groups at Risoe who conduct basic research or technological R and D. This division employs a group of scientific electronics consultants, a production group, a maintenance facility and a central instrument pool for the complete site. The other division, whose work is reported here, is engaged in both basic and applied research in topics and areas of direct interest to the department itself. These involve the development and evaluation of methods, for example, in the field of plant reliability and safety as well as in a group engaged in nuclear geophysical activities - including mineral prospecting in Greenland. Another team is working with man-machine problems in highly automated systems and the associated needs for computer support in connection with proper job allocation and design. Finally, a group is active in the field of applied laser physics. The time period covered is 1975-1977. (author/BP)
International Nuclear Information System (INIS)
Mankuzhiyil, Nijil; Ansoldi, Stefano; Persic, Massimo; Tavecchio, Fabrizio
2011-01-01
For the high-frequency-peaked BL Lac object Mrk 421, we study the variation of the spectral energy distribution (SED) as a function of source activity, from quiescent to active. We use a fully automatized χ 2 -minimization procedure, instead of the 'eyeball' procedure more commonly used in the literature, to model nine SED data sets with a one-zone synchrotron self-Compton (SSC) model and examine how the model parameters vary with source activity. The latter issue can finally be addressed now, because simultaneous broadband SEDs (spanning from optical to very high energy photon) have finally become available. Our results suggest that in Mrk 421 the magnetic field (B) decreases with source activity, whereas the electron spectrum's break energy (γ br ) and the Doppler factor (δ) increase-the other SSC parameters turn out to be uncorrelated with source activity. In the SSC framework, these results are interpreted in a picture where the synchrotron power and peak frequency remain constant with varying source activity, through a combination of decreasing magnetic field and increasing number density of γ ≤ γ br electrons: since this leads to an increased electron-photon scattering efficiency, the resulting Compton power increases, and so does the total (= synchrotron plus Compton) emission.
Active Cooling and Thermal Management of a Downhole Tool Electronics Section
DEFF Research Database (Denmark)
Soprani, Stefano; Engelbrecht, Kurt; Just Nørgaard, Anders
2015-01-01
combines active and passive cooling techniques, aiming at an efficient thermal management, preserving the tool compactness and avoiding the use of moving parts. Thermoelectric coolers were used to transfer the dissipated heat from the temperature-sensitive electronics to the external environment. Thermal...... contact resistances were minimized and thermally insulating foam protected the refrigerated microenvironment from the hot surroundings....
One size fits all electronics for insole-based activity monitoring.
Hegde, Nagaraj; Bries, Matthew; Melanson, Edward; Sazonov, Edward
2017-07-01
Footwear based wearable sensors are becoming prominent in many areas of monitoring health and wellness, such as gait and activity monitoring. In our previous research we introduced an insole based wearable system SmartStep, which is completely integrated in a socially acceptable package. From a manufacturing perspective, SmartStep's electronics had to be custom made for each shoe size, greatly complicating the manufacturing process. In this work we explore the possibility of making a universal electronics platform for SmartStep - SmartStep 3.0, which can be used in the most common insole sizes without modifications. A pilot human subject experiments were run to compare the accuracy between the one-size fits all (SmartStep 3.0) and custom size SmartStep 2.0. A total of ~10 hours of data was collected in the pilot study involving three participants performing different activities of daily living while wearing SmartStep 2.0 and SmartStep 3.0. Leave one out cross validation resulted in a 98.5% average accuracy from SmartStep 2.0, while SmartStep 3.0 resulted in 98.3% accuracy, suggesting that the SmartStep 3.0 can be as accurate as SmartStep 2.0, while fitting most common shoe sizes.
Directory of Open Access Journals (Sweden)
Jennifer Vandooren
Full Text Available Gelatinase B or matrix metalloproteinase-9 (MMP-9 (EC 3.4.24.35 is increased in inflammatory processes and cancer, and is associated with disease progression. In part, this is due to MMP-9-mediated degradation of extracellular matrix, facilitating influx of leukocytes into inflamed tissues and invasion or metastasis of cancer cells. MMP-9 is produced as proMMP-9 and its propeptide is subsequently removed by other proteases to generate proteolytically active MMP-9. The significance of MMP-9 in pathologies triggered the development of specific inhibitors of this protease. However, clinical trials with synthetic inhibitors of MMPs in the fight against cancer were disappointing. Reports on active compounds which inhibit MMP-9 should be carefully examined in this regard. In a considerable set of recent publications, two antibiotics (minocycline and azythromycin and the proteasome inhibitor bortezomib, used in cancers, were reported to inhibit MMP-9 at different stages of its expression, activation or activity. The current study was undertaken to compare and to verify the impact of these compounds on MMP-9. With exception of minocycline at high concentrations (>100 μM, the compounds did not affect processing of proMMP-9 into MMP-9, nor did they affect direct MMP-9 gelatinolytic activity. In contrast, azithromycin specifically reduced MMP-9 mRNA and protein levels without affecting NF-κB in endotoxin-challenged monocytic THP-1 cells. Bortezomib, although being highly toxic, had no MMP-9-specific effects but significantly upregulated cyclooxygenase-2 (COX-2 activity and PGE2 levels. Overall, our study clarified that azithromycin decreased the levels of MMP-9 by reduction of gene and protein expression while minocycline inhibits proteolytic activity at high concentrations.
Electronic Commerce and Electronic Business
Indian Academy of Sciences (India)
R. Narasimhan (Krishtel eMaging) 1461 1996 Oct 15 13:05:22
This special issue is motivated by the recent upsurge of research activity in the areas of electronic commerce and electronic business both in India and all over the world. The current ... Monte Carlo methods for pricing financial options are then.
Mainz, Emilie R; Serafin, D Stephen; Nguyen, Tuong T; Tarrant, Teresa K; Sims, Christopher E; Allbritton, Nancy L
2016-08-02
The etiology of rheumatoid arthritis (RA) is poorly understood, and 30% of patients are unresponsive to established treatments targeting tumor necrosis factor α (TNFα). Akt kinase is implicated in TNFα signaling and may act as a barometer of patient responses to biologic therapies. Fluorescent peptide sensors and chemical cytometry were employed to directly measure Akt activity as well as proteolytic activity in individual fibroblast-like synoviocytes (FLS) from RA and normal subjects. The specificity of the peptide reporter was evaluated and shown to be a valid measure of Akt activity in single cells. The effect of TNFα treatment on Akt activity was highly heterogeneous between normal and RA subjects, which was not observable in bulk analyses. In 2 RA subjects, a bimodal distribution of Akt activity was observed, primarily due to a subpopulation (21.7%: RA Subject 5; 23.8%: RA Subject 6) of cells in which >60% of the reporter was phosphorylated. These subjects also possessed statistically elevated proteolytic cleavage of the reporter relative to normal subjects, suggesting heterogeneity in Akt and protease activity that may play a role in the RA-affected joint. We expect that chemical cytometry studies pairing peptide reporters with capillary electrophoresis will provide valuable data regarding aberrant kinase activity from small samples of clinical interest.
Sawtooth activity of the ion cloud in an electron-beam ion trap
International Nuclear Information System (INIS)
Radtke, R.; Biedermann, C.
2003-01-01
The dynamics of an ensemble of highly charged Ar and Ba ions in an electron-beam ion trap (EBIT) was studied by recording time-resolved x-ray spectra emitted from trapped ions. Sawtoothlike signatures manifest in the spectra for a variety of EBIT operating conditions indicating a sudden collapse of the ion inventory in the trap. The collapse occurs on a time scale of approximately 100 ms and the evolution of the sawteeth is very sensitive to parameters such as electron-beam current and axial trap depth. Analysis of the measurements is based on a time-dependent calculation of the trapping process showing that sawtooth activity is caused by the feedback between the low-Z argon and high-Z barium ions. This unexpected behavior demonstrates the importance of nonlinear effects in electron-beam traps containing more than a single ion species
Beutner, Gisela; Eliseev, Roman A; Porter, George A
2014-01-01
Mitochondria provide energy in form of ATP in eukaryotic cells. However, it is not known when, during embryonic cardiac development, mitochondria become able to fulfill this function. To assess this, we measured mitochondrial oxygen consumption and the activity of the complexes (Cx) 1 and 2 of the electron transport chain (ETC) and used immunoprecipitation to follow the generation of mitochondrial supercomplexes. We show that in the heart of mouse embryos at embryonic day (E) 9.5, mitochondrial ETC activity and oxidative phosphorylation (OXPHOS) are not coupled, even though the complexes are present. We show that Cx-1 of the ETC is able to accept electrons from the Krebs cycle, but enzyme assays that specifically measure electron flow to ubiquinone or Cx-3 show no activity at this early embryonic stage. At E11.5, mitochondria appear functionally more mature; ETC activity and OXPHOS are coupled and respond to ETC inhibitors. In addition, the assembly of highly efficient respiratory supercomplexes containing Cx-1, -3, and -4, ubiquinone, and cytochrome c begins at E11.5, the exact time when Cx-1 becomes functional activated. At E13.5, ETC activity and OXPHOS of embryonic heart mitochondria are indistinguishable from adult mitochondria. In summary, our data suggest that between E9.5 and E11.5 dramatic changes occur in the mitochondria of the embryonic heart, which result in an increase in OXPHOS due to the activation of complex 1 and the formation of supercomplexes.
Energy Technology Data Exchange (ETDEWEB)
Shi, Ming W.; Stewart, Scott G.; Sobolev, Alexandre N.; Dittrich, Birger; Schirmeister, Tanja; Luger, Peter; Hesse, Malte; Chen, Yu-Sheng; Spackman, Peter R.; Spackman, Mark A.; Grabowsky, Simon (Heinrich-Heine); (Freie); (UC); (Bremen); (JG-UM); (UWA)
2017-01-24
The trans-epoxysuccinyl amide group as a biologically active moiety in cysteine protease inhibitors such as loxistatin acid E64c has been used as a benchmark system for theoretical studies of environmental effects on the electron density of small active ingredients in relation to their biological activity. Here, the synthesis and the electronic properties of the smallest possible active site model compound are reported to close the gap between the unknown experimental electron density of trans-epoxysuccinyl amides and the well-known function of related drugs. Intramolecular substituent effects are separated from intermolecular crystal packing effects on the electron density, which allows us to predict the conditions under which an experimental electron density investigation on trans-epoxysuccinyl amides will be possible. In this context, the special importance of the carboxylic acid function in the model compound for both crystal packing and biological activity is revealed through the novel tool of model energy analysis.
Development of microalgae biomaterials with enhanced antioxidant activity using electron beam
Energy Technology Data Exchange (ETDEWEB)
Kim, Younghwa; Park, Hyunjin; Choi, Soojeong; Lee, Jaehwa [Silla Univ., Busan (Korea, Republic of)
2013-07-01
By increasing the antioxidant products (e. g. antioxidant enzyme, carotenoid, phycobiliproteins, chlorophyll, lipid phenolic compounds, etc.) in microalgae, it could be useful for industry. In this study, mutants of fresh water microalgae Arthrospira platensis (A. platensis) by high energy electron beam were isolated and characterized. Those selected mutants showed higher growth rate than parental strain. The antioxidant enzyme activity (SOD and POD), flavonoid, phenolic compound and phycocyanin of mutants were increased about 2 times compared to wild type. Moreover, DPPH radical scavenging activity was increased about 20%. Microalgae species with improved growth rate and enhanced active compounds make the commercial process more feasible in industry. Using microalgae mutants with increased antioxidant products, it is useful to develop microalgae biomaterials for neutraceuticals.
Development of microalgae biomaterials with enhanced antioxidant activity using electron beam
International Nuclear Information System (INIS)
Kim, Younghwa; Park, Hyunjin; Choi, Soojeong; Lee, Jaehwa
2013-01-01
By increasing the antioxidant products (e. g. antioxidant enzyme, carotenoid, phycobiliproteins, chlorophyll, lipid phenolic compounds, etc.) in microalgae, it could be useful for industry. In this study, mutants of fresh water microalgae Arthrospira platensis (A. platensis) by high energy electron beam were isolated and characterized. Those selected mutants showed higher growth rate than parental strain. The antioxidant enzyme activity (SOD and POD), flavonoid, phenolic compound and phycocyanin of mutants were increased about 2 times compared to wild type. Moreover, DPPH radical scavenging activity was increased about 20%. Microalgae species with improved growth rate and enhanced active compounds make the commercial process more feasible in industry. Using microalgae mutants with increased antioxidant products, it is useful to develop microalgae biomaterials for neutraceuticals
Spoilage potential of Pseudomonas species isolated from goat milk.
Scatamburlo, T M; Yamazi, A K; Cavicchioli, V Q; Pieri, F A; Nero, L A
2015-02-01
Pseudomonas spp. are usually associated with spoilage microflora of dairy products due to their proteolytic potential. This is of particular concern for protein-based products, such as goat milk cheeses and fermented milks. Therefore, the goal of the present study was to characterize the proteolytic activity of Pseudomonas spp. isolated from goat milk. Goat milk samples (n=61) were obtained directly from bulk tanks on dairy goat farms (n=12), and subjected to a modified International Organization for Standardization (ISO) protocol to determine the number and proteolytic activity of Pseudomonas spp. Isolates (n=82) were obtained, identified by PCR, and subjected to pulsed-field gel electrophoresis with XbaI macro-restriction. Then, the isolates were subjected to PCR to detect the alkaline protease gene (apr), and phenotypic tests were performed to check proteolytic activity at 7°C, 25°C, and 35°C. Mean Pseudomonas spp. counts ranged from 2.9 to 4.8 log cfu/mL, and proteolytic Pseudomonas spp. counts ranged from 1.9 to 4.6 log cfu/mL. All isolates were confirmed to be Pseudomonas spp., and 41 were identified as Pseudomonas fluorescens, which clustered into 5 groups sharing approximately 82% similarity. Thirty-six isolates (46.9%) were positive for the apr gene; and 57 (69.5%) isolates presented proteolytic activity at 7°C, 82 (100%) at 25°C, and 64 (78%) at 35°C. The isolates were distributed ubiquitously in the goat farms, and no relationship among isolates was observed when the goat farms, presence of apr, pulsotypes, and proteolytic activity were taken into account. We demonstrated proteolytic activity of Pseudomonas spp. present in goat milk by phenotypic and genotypic tests and indicated their spoilage potential at distinct temperatures. Based on these findings and the ubiquity of Pseudomonas spp. in goat farm environments, proper monitoring and control of Pseudomonas spp. during production are critical. Copyright © 2015 American Dairy Science Association
Modelling of electron transport and of sawtooth activity in tokamaks
International Nuclear Information System (INIS)
Angioni, C.
2001-10-01
Transport phenomena in tokamak plasmas strongly limit the particle and energy confinement and represent a crucial obstacle to controlled thermonuclear fusion. Within the vast framework of transport studies, three topics have been tackled in the present thesis: first, the computation of neoclassical transport coefficients for general axisymmetric equilibria and arbitrary collisionality regime; second, the analysis of the electron temperature behaviour and transport modelling of plasma discharges in the Tokamak a configuration Variable (TCV); third, the modelling and simulation of the sawtooth activity with different plasma heating conditions. The work dedicated to neoclassical theory has been undertaken in order to first analytically identify a set of equations suited for implementation in existing Fokker-Planck codes. Modifications of these codes enabled us to compute the neoclassical transport coefficients considering different realistic magnetic equilibrium configurations and covering a large range of variation of three key parameters: aspect ratio, collisionality, and effective charge number. A comparison of the numerical results with an analytical limit has permitted the identification of two expressions for the trapped particle fraction, capable of encapsulating the geometrical effects and thus enabling each transport coefficient to be fitted with a single analytical function. This has allowed us to provide simple analytical formulae for all the neoclassical transport coefficients valid for arbitrary aspect ratio and collisionality in general realistic geometry. This work is particularly useful for a correct evaluation of the neoclassical contribution in tokamak scenarios with large bootstrap cur- rent fraction, or improved confinement regimes with low anomalous transport and for the determination of the plasma current density profile, since the plasma conductivity is usually assumed neoclassical. These results have been included in the plasma transport code
PAPP-A proteolytic activity enhances IGF bioactivity in ascites from women with ovarian carcinoma
DEFF Research Database (Denmark)
Thomsen, Jacob; Hjortebjerg, Rikke; Espelund, Ulrick
2015-01-01
Pregnancy-associated plasma protein-A (PAPP-A) stimulates insulin-like growth factor (IGF) action through proteolysis of IGF-binding protein (IGFBP)-4. In experimental animals, PAPP-A accelerates ovarian tumor growth by this mechanism. To investigate the effect of PAPP-A in humans, we compared.......03). Ascites was more potent than serum in activating the IGF-I receptor (IGF-IR) in vitro (+31%, P IGF-I, and lower levels of IGF-II (P ... of IGF-IR in all but one tumor, whereas all tumors expressed PAPP-A, IGFBP-4, IGF-I and IGF-II. Addition of recombinant PAPP-A to ascites increased the cleavage of IGFBP-4 and enhanced IGF-IR activation (P IGFs and these proteins...
International Nuclear Information System (INIS)
Klyachkin, B.M.; Basargin, S.T.; Timofeev, I.V.; Khaliulin, Yu.G.; Dorofeev, S.A.; Alekseenko, L.D.; Gumenyuk, M.L.
1992-01-01
The study of the effect of sodium mefenaminate on radiation resistance of mice yielded positive results. Clinical investigations showed mefenamic acid to decrease the activity of cathepsin D-like protease in colonic cancer tissue. The acid field to affect the proteolytic activity of the normal mucosa. It revealed an immunomodulating activity and influenced the hemostatic system which usually manifested itself in amelioration of hypercoagulation
Kurimoto, Naoya; Omoda, Ryo; Mizumo, Tomonobu; Ito, Seitaro; Aihara, Yuichi; Itoh, Takahito
2018-02-01
Quinoid compounds are important candidates of organic active materials for lithium-ion batteries. However, its high solubility to organic electrolyte solutions and low redox potential are known as their major drawbacks. To circumvent these issues, we have designed and synthesized a tandem-tetracyanoquinonedimethane type cathode-active material, 11,11,12,12,13,13,14,14-octacyano-1,4,5,8-anthradiquinotetramethane (OCNAQ), that has four redox sites per molecule, high redox potential and suppressed solubility to electrolyte solution. Synthesized OCNAQ has been found to have two-step redox reactions by cyclic voltammetry, and each step consists of two-electron reactions. During charge-discharge tests using selected organic cathode-active materials with a lithium metal anode, the cell voltages obtained from OCNAQ are higher than those for 11,11-dicyanoanthraquinone methide (AQM) as expected, due to the strong electron-withdrawing effect of the cyano groups. Unfortunately, even with the use of the organic active material, the issue of dissolution to the electrolyte solution cannot be suppressed completely; however, appropriate choice of the electrolyte solutions, glyme-based electrolyte solutions in this study, give considerable improvement of the cycle retention (98% and 56% at 10 and 100 cycles at 0.5C, respectively). The specific capacity and energy density obtained in this study are 206 mAh g-1 and 554 mWh g-1 with respect to the cathode active material.
Effects of proteolysis on the adenosinetriphosphatase activities of thymus myosin
International Nuclear Information System (INIS)
Vu, N.D.; Wagner, P.D.
1987-01-01
Limited proteolysis was used to identify regions on the heavy chains of calf thymus myosin which may be involved in ATP and actin binding. Assignments of the various proteolytic fragments to different parts of the myosin heavy chain were based on solubility, gel filtration, electron microscopy, and binding of 32 P-labeled regulatory light chains. Chymotrypsin rapidly cleaved within the head of thymus myosin to give a 70,000-dalton N-terminal fragment and a 140,000-dalton C-terminal fragment. These two fragments did not dissociate under nondenaturing conditions. Cleavage within the myosin tail to give heavy meromyosin occurred more slowly. Cleavage at the site 70,000 daltons from the N-terminus of the heavy chain caused about a 30-fold decrease in the actin concentration required to achieve half-maximal stimulation of the magnesium-adenosinetriphosphatase (Mg-ATPase) activity of unphosphorylated thymus myosin. The actin-activated ATPase activity of this digested myosin was only slightly affected by light chain phosphorylation. Actin inhibited the cleavage at this site by chymotrypsin. In the presence of ATP, chymotrypsin rapidly cleaved the thymus myosin heavy chain at an additional site about 4000 daltons from the N-terminus. Cleavage at this site caused a 2-fold increase in the ethylenediaminetetraacetic acid-ATPase activity and 3-fold decreases in the Ca 2+ - and Mg-ATPase activities of thymus myosin. Thus, cleavage at the N-terminus of thymus myosin was affected by ATP, and this cleavage altered ATPase activity. Papain cleaved the thymus myosin heavy chain about 94,000 daltons from the N-terminus to give subfragment 1. Although this subfragment 1 contained intact light chains, its actin-activated ATPase activity was not affected by light chain phosphorylation
Kooij, A.; Frederiks, W. M.; Gossrau, R.; van Noorden, C. J.
1991-01-01
We have detected xanthine oxidoreductase activity in unfixed cryostat sections of rat and chicken liver, rat duodenum, and bovine mammary gland using the tissue protectant polyvinyl alcohol, the electron carrier 1-methoxyphenazine methosulfate, the final electron acceptor Tetranitro BT, and
Directory of Open Access Journals (Sweden)
Rita Costa
Full Text Available Alzheimer's disease (AD is a neurodegenerative disorder characterized by the deposition of amyloid beta-peptide (A-Beta in the brain. Transthyretin (TTR is a tetrameric protein of about 55 kDa mainly produced in the liver and choroid plexus of the brain. The known physiological functions of TTR are the transport of thyroid hormone T(4 and retinol, through binding to the retinol binding protein. TTR has also been established as a cryptic protease able to cleave ApoA-I in vitro. It has been described that TTR is involved in preventing A-Beta fibrilization, both by inhibiting and disrupting A-Beta fibrils, with consequent abrogation of toxicity. We further characterized the nature of the TTR/A-Beta interaction and found that TTR, both recombinant or isolated from human sera, was able to proteolytically process A-Beta, cleaving the peptide after aminoacid residues 1, 2, 3, 10, 13, 14,16, 19 and 27, as determined by mass spectrometry, and reversed phase chromatography followed by N-terminal sequencing. A-Beta peptides (1-14 and (15-42 showed lower amyloidogenic potential than the full length counterpart, as assessed by thioflavin binding assay and ultrastructural analysis by transmission electron microscopy. A-Beta cleavage by TTR was inhibited in the presence of an alphaAPP peptide containing the Kunitz Protease Inhibitor (KPI domain but not in the presence of the secreted alphaAPP derived from the APP isoform 695 without the KPI domain. TTR was also able to degrade aggregated forms of A-Beta peptide. Our results confirmed TTR as a protective molecule in AD, and prompted A-Beta proteolysis by TTR as a protective mechanism in this disease. TTR may prove to be a useful therapeutic agent for preventing or retarding the cerebral amyloid plaque formation implicated in AD pathology.
International Nuclear Information System (INIS)
Sampa, Maria Helena de O.; Rela, Paulo R.; Duarte, Celina Lopes; Las Casas, Alexandre; Mori, Manoel Nunes; Omi, Nelson M.
2005-01-01
A technical and economical feasibility study was performed comparing the use electron beam and activated charcoal for treatment of industrial wastewater. In this study was used synthetic solutions, prepared in laboratory with organic compounds standards, where the composition was focused on the critical organic contaminants usually presented in wastewater from petrochemical industry. For the sample irradiation was used an industrial electron beam from Radiation Dynamics Inc. 1.5 MeV - 37.5 kW setup in IPEN. The doses ranged from 5 kGy to 100 kGy. A common granulated activated charcoal in a fixed-bed absorber glass column was used to study the pollutants absorption performance. The results show that if the adequate irradiation dose was delivered to the organic pollutant, it is possible to conclude for the studied compounds that the Electron Beam Process is, in aspect of organic removal efficiency, similar to the activated carbon process. (author)
International Nuclear Information System (INIS)
Cabrera, M.A.; Ezquer, R.G.; Mosert, M.; Jadur, C.A.
2002-01-01
The International Reference Ionosphere model only gives the vertical electron content (VTEC). In this paper the slant electron content (SEC) for the ATS 6 satellite - Palehua (21.4 deg. N, 201.9 deg. E) radio signal path for a middle solar activity year is calculated. To this end, IRI model is used to obtain the electron density at different points of the signal path. Equinoxes and solstices are considered. Measurements obtained with Faraday rotation technique at Palehua are compared with the modelled values. Although overestimation was observed for night hours, the results show good SEC predictions for several hours at period of maximum ionisation, suggesting that would be possible to model the STEC using IRI. (author)
Effect of electron-ion treatment on fermentative activity of food yeasts
International Nuclear Information System (INIS)
Sergeev, I.N.; Ostapenkov, A.M.
1988-01-01
Investigation into effect of electron-ion treatment (EIT) on fermentative activity (FA) of Sacch cerevisial type yeasts of 12 breed was conducted. It is shown that even within the limits of one and the same type different treatment regimes are meded. This is obviously connected with physiologic peculiarities of different yeast breeds. Therefore an individual optimal treatment regime should be determined in each particular case during EIT of different breeds
Directory of Open Access Journals (Sweden)
Piek Jan P
2009-06-01
Full Text Available Abstract Background Many children are reported to have insufficient physical activity (PA placing them at greater risk of poor health outcomes. Participating in sedentary activities such as playing electronic games is widely believed to contribute to less PA. However there is no experimental evidence that playing electronic games reduces PA. There is also no evidence regarding the effect of different types of electronic games (traditional sedentary electronic games versus new active input electronic games on PA. Further, there is a poor understanding about how characteristics of children may moderate the impact of electronic game access on PA and about what leisure activities are displaced when children play electronic games. Given that many children play electronic games, a better understanding of the effect of electronic game use on PA is critical to inform child health policy and intervention. Methods This randomised and controlled trial will examine whether PA is decreased by access to electronic games and whether any effect is dependent on the type of game input or the child's characteristics. Children aged 10–12 years (N = 72, 36 females will be recruited and randomised to a balanced ordering of 'no electronic games', 'traditional' electronic games and 'active' electronic games. Each child will participate in each condition for 8 weeks, and be assessed prior to participation and at the end of each condition. The primary outcome is PA, assessed by Actical accelerometers worn for 7 days on the wrist and hip. Energy expenditure will be assessed by the doubly labelled water technique and motor coordination, adiposity, self-confidence, attitudes to technology and PA and leisure activities will also be assessed. A sample of 72 will provide a power of > 0.9 for detecting a 15 mins difference in PA (sd = 30 mins. Discussion This is the first such trial and will provide critical information to understand whether access to electronic games affects
Protease-activated quantum dot probes
International Nuclear Information System (INIS)
Chang, Emmanuel; Miller, Jordan S.; Sun, Jiantang; Yu, William W.; Colvin, Vicki L.; Drezek, Rebekah; West, Jennifer L.
2005-01-01
We have developed a novel nanoparticulate luminescent probe with inherent signal amplification upon interaction with a targeted proteolytic enzyme. This construct may be useful for imaging in cancer detection and diagnosis. In this system, quantum dots (QDs) are bound to gold nanoparticles (AuNPs) via a proteolytically degradable peptide sequence to non-radiatively suppress luminescence. A 71% reduction in luminescence was achieved with conjugation of AuNPs to QDs. Release of AuNPs by peptide cleavage restores radiative QD photoluminescence. Initial studies observed a 52% rise in luminescence over 47 h of exposure to 0.2 mg/mL collagenase. These probes can be customized for targeted degradation simply by changing the sequence of the peptide linker
Heinen, André; Aldakkak, Mohammed; Stowe, David F.; Rhodes, Samhita S.; Riess, Matthias L.; Varadarajan, Srinivasan G.; Camara, Amadou K. S.
2007-01-01
Mitochondria generate reactive oxygen species (ROS) dependent on substrate conditions, O(2) concentration, redox state, and activity of the mitochondrial complexes. It is well known that the FADH(2)-linked substrate succinate induces reverse electron flow to complex I of the electron transport chain
Goeminne, Pieter C; Vandooren, Jennifer; Moelants, Eva A; Decraene, Ann; Rabaey, Evelyn; Pauwels, Ans; Seys, Sven; Opdenakker, Ghislain; Proost, Paul; Dupont, Lieven J
2014-02-01
Non-cystic fibrosis bronchiectasis (NCFB) is characterized by a vicious cycle of airway infection, inflammation and structural damage with inappropriate mucus clearance. Our aim was to relate the value of proteolytic enzymes, proteolytic enzyme activity and inflammatory markers to disease severity and symptoms in patients with NCFB. Sputum induction in NCFB patients and healthy controls was performed. Sputum was analysed for total and differential cell count, markers of inflammation (CXCL8 (also known as interleukin-8) and tumour necrosis factor-α (TNF-α)) and proteolytic enzymes (neutrophil elastase (NE), gelatin zymography and total gelatinolytic activity (TGA)). Each patient was evaluated by spirometry, Leicester Cough Questionnaire (LCQ) and Sputum Colour Chart (SCC). Patient files were analysed to determine Pseudomonas aeruginosa colonization status. The computed tomography (CT) closest to the date sputum induction was scored by a radiologist. NCFB patients showed significantly higher neutrophils, CXCL8, TNF-α, NE and TGA than healthy controls. TGA subanalysis showed that the majority of the activity was NE (82 ± 6.4%). Residual activity was mainly zinc ion-dependent matrix metalloproteinase (MMP) activity (18 ± 6.4%). Subanalysis showed that patients with chronic Pseudomonas aeruginosa colonization had more activated MMP-9. Correlations were seen between proteolytic enzymes and inflammation and disease severity (spirometry and CT score), but not with the LCQ. SCC was associated with increased markers of inflammation, proteolytic enzymes and worse CT score. We show that sputum purulence assessment in daily clinical practice using the SCC is a quick and easy tool that reflects severity of inflammation, destruction and proteolytic enzymatic activity/presence. © 2013 The Authors. Respirology © 2013 Asian Pacific Society of Respirology.
Hinkley, Trina; Timperio, Anna; Salmon, Jo; Hesketh, Kylie
2017-04-01
Little is known about the associations of preschoolers' health behaviors with their later psychosocial wellbeing. This study investigates the association of 3- to 5-year-old children's physical activity and electronic media use with their later social-emotional skills (6-8 years). Data were collected in 2008-2009 and 2011-2012 for the Healthy Active Preschool and Primary Years (HAPPY) Study in metropolitan Melbourne. Participants were a random subsample (n = 108) of the 567 children at follow-up. Physical activity was objectively measured using ActiGraph GT1M accelerometers; electronic media use (television viewing, sedentary electronic games and active electronic games) was parent proxy-reported. Social and emotional skills were child-reported using the Bar-On Emotional Quotient Inventory-Youth Version. Regression analyses controlled for sex, clustering by center of recruitment, and accelerometer wear time (for physical activity analyses). Sedentary electronic games were positively associated with intrapersonal and stress management skills and total emotional quotient. Computer/internet use was inversely associated with interpersonal, and positively associated with stress management, skills. Findings suggest that physical activity is not associated with children's psychosocial health while some types of electronic media use are. Future research should investigate the contexts in which preschoolers participate in these behaviors and potential causal mechanisms of associations.
Ramiprilate inhibits functional matrix metalloproteinase activity in Crohn's disease fistulas
DEFF Research Database (Denmark)
Efsen, Eva; Saermark, Torben; Hansen, Alastair
2011-01-01
Increased expression of matrix metalloproteinase (MMP)-2, -3 and -9 has been demonstrated in Crohn's disease fistulas, but it is unknown whether these enzymes are biologically active and represent a therapeutic target. Therefore, we investigated the proteolytic activity of MMPs in fistula tissue...... from six controls were also included. Total functional MMP activity was measured by a high-pressure liquid chromatography (HPLC)-based, fluorogenic MMP-substrate cleavage assay, and the specific activity of MMP-2, -3 and -9 by the MMP Biotrak Activity Assay. The MMP inhibitors comprised ethylene......-9.83) compared with non-Crohn's fistulas, [0.32 ng/ml, range 0-2.66, (p MMP-9 activity [0.64 ng/ml, range 0-5.66 and 0.17 ng/ml, range 0-1.1, respectively (p MMP activity level by 42% and suppressed the specific MMP-3...
Electronic Nose Technology to Measure Soil Microbial Activity and Classify Soil Metabolic Status
Fabrizio De Cesare; Elena Di Mattia; Simone Pantalei; Emiliano Zampetti; Vittorio Vinciguerra; Antonella Macagnano
2011-01-01
The electronic nose (E-nose) is a sensing technology that has been widely used to monitor environments in the last decade. In the present study, the capability of an E-nose, in combination with biochemical and microbiological techniques, of both detecting the microbial activity and estimating the metabolic status of soil ecosystems, was tested by measuring on one side respiration, enzyme activities and growth of bacteria in natural but simplified soil ecosystems over 23 days of incubation thr...
Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.
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
Meng, Tian; Kou, Zongkui; Amiinu, Ibrahim Saana; Hong, Xufeng; Li, Qingwei; Tang, Yongfu; Zhao, Yufeng; Liu, Shaojun; Mai, Liqiang; Mu, Shichun
2018-04-17
Tuning the electron structure is of vital importance for designing high active electrode materials. Here, for boosting the capacitive performance of tungsten oxide, an atomic scale engineering approach to optimize the electronic structure of tungsten oxide by Ni doping is reported. Density functional theory calculations disclose that through Ni doping, the density of state at Fermi level for tungsten oxide can be enhanced, thus promoting its electron transfer. When used as electrode of supercapacitors, the obtained Ni-doped tungsten oxide with 4.21 at% Ni exhibits an ultrahigh mass-specific capacitance of 557 F g -1 at the current density of 1 A g -1 and preferable durability in a long-term cycle test. To the best of knowledge, this is the highest supercapacitor performance reported so far in tungsten oxide and its composites. The present strategy demonstrates the validity of the electronic structure control in tungsten oxide via introducing Ni atoms for pseudocapacitors, which can be extended to other related fields as well. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Kannan, Ramakrishnan; Cox, Eric; Wang, Lei; Kuzina, Irina; Gu, Qun; Giniger, Edward
2018-01-17
Notch signaling is required for the development and physiology of nearly every tissue in metazoans. Much of Notch signaling is mediated by transcriptional regulation of downstream target genes, but Notch controls axon patterning in Drosophila by local modulation of Abl tyrosine kinase signaling, via direct interactions with the Abl co-factors Disabled and Trio. Here, we show that Notch-Abl axonal signaling requires both of the proteolytic cleavage events that initiate canonical Notch signaling. We further show that some Notch protein is tyrosine phosphorylated in Drosophila , that this form of the protein is selectively associated with Disabled and Trio, and that relevant tyrosines are essential for Notch-dependent axon patterning but not for canonical Notch-dependent regulation of cell fate. Based on these data, we propose a model for the molecular mechanism by which Notch controls Abl signaling in Drosophila axons. © 2018. Published by The Company of Biologists Ltd.
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.
Low temperature electron beam irradiation effects on the lactate dehydrogenase activity
International Nuclear Information System (INIS)
Catana, D.; Hategan, Alina; Oproiu, C.; Popescu, Alina; Hategan, Dora; Morariu, V. V.
1998-01-01
The direct and indirect effects of 5 MeV electron beam irradiation in the range 0-400 Gy at 20 deg. C, -3 deg. C and -196 deg. C on the global enzymatic activity of lactate dehydrogenase (LDH) have been studied. Our results showed a monoexponential decrease in the enzymatic activity of irradiated LDH at all irradiation temperatures independently of direct or indirect action of radiation. The temperature gradient used to lower the temperature of the samples to -196 deg. C drastically influences the results. Our data suggest that freeze-thawing in two steps down to -196 deg. C make LDH insensitive to irradiation, while one step freeze-thawing procedure results in a gradual activity loss with increasing dose irradiation. This data can be interpreted in terms of different conformational changes during the particular freeze-thawing process. (authors)
Effects of cysteine protease inhibitors on rabbit cathepsin D maturation
International Nuclear Information System (INIS)
Samarel, A.M.; Ferguson, A.G.; Decker, R.S.; Lesch, M.
1989-01-01
To examine the effects of cysteine protease inhibitors on cathepsin D intracellular transport, proteolytic processing, and secretion, primary cultures of rabbit cardiac fibroblasts were grown to confluence and exposed to media containing leupeptin, E 64, or chloroquine. Cathepsin D maturation was then evaluated in pulse-chase biosynthetic labeling experiments. None of the three agents affected the charge modification of procathepsin D within the Golgi apparatus. However, all three agents interfered with the subsequent proteolytic processing of procathepsin D isoforms to active cathepsin D. Both leupeptin and E 64 caused the intracellular accumulation of large amounts of a Mr 51,000 processing intermediate. Trace amounts of this intermediate were also detected in chloroquine-treated cells. Combined activity assay and radioimmunoassay of cell lysates indicated that this partially processed form of cathepsin D possessed proteolytic activity. Whereas low medium concentrations of leupeptin (10-100 microM) but not E 64 appeared to stimulate procathepsin D secretion, neither agent appeared to have a major effect on the rate of proenzyme secretion at doses required to inhibit proteolytic maturation (1-10 mM). Furthermore, pretreatment of cells with 10 mM leupeptin appeared only to delay, but not prevent, the intracellular transport of cathepsin D to lysosomes. In contrast, chloroquine increased procathepsin D secretion in a dose-dependent manner, diverting the majority of newly synthesized procathepsin D from the intracellular protease(s) responsible for proteolytic processing. These results suggest that cysteine proteases participate in the proteolytic maturation of procathepsin D during the transport of newly synthesized enzyme to lysosomes, but cysteine protease-mediated proteolytic processing is not required for cathepsin D activation or lysosomal translocation
Enhanced activity of meprin-α, a pro-migratory and pro-angiogenic protease, in colorectal cancer.
Directory of Open Access Journals (Sweden)
Daniel Lottaz
Full Text Available Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF-induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer.
Intervention with Serine Protease Activity with Small Peptides
DEFF Research Database (Denmark)
Xu, Peng
2015-01-01
Serine proteases perform proteolytic reactions in many physiological and metabolic processes and have been certified as targets for therapeutics. Small peptides can be used as potent antagonists to target serine proteases and intervene with their activities. Urokinase-type plasminogen activator (u......PA) plays an important role in plasminogen activation system, which has many physiological and pathological functions and is closely associated with the metastasis of tumor cells. Based on a mono-cyclic peptidic inhibitor of murine uPA (muPA), mupain-1, which was screened out from a phage-display library...... before, we elucidated the binding and inhibitory mechanism by using multiple techniques, like X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and surface plasmon resonance analysis. By studying the peptide-enzyme interaction, we discovered an unusual inhibitor...
Directory of Open Access Journals (Sweden)
Lin PY
2015-10-01
Full Text Available Pao-Yen Lin1,2 1Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, 2Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, Taiwan Abstract: Evidence has supported the role of brain-derived neurotrophic factor (BDNF in antidepressant effect. The precursor of BDNF (proBDNF often exerts opposing biological effects on mature BDNF (mBDNF. Hence, the balance between proBDNF and mBDNF might be critical in total neurotrophic effects, leading to susceptibility to or recovery from depression. In the current study, we measured the protein expression levels of proBDNF, and its proteolytic products, truncated BDNF, and mBDNF, in human SH-SY5Y cells treated with different antidepressants. We found that the treatment significantly increased the production of mBDNF, but decreased the production of truncated BDNF and proBDNF. These results support that antidepressants can promote proBDNF cleavage. Further studies are needed to clarify whether proBDNF cleavage plays a role in antidepressant mechanisms. Keywords: antidepressant, mature BDNF, neurotrophic effect, proBDNF cleavage
Bryant, M L; Ratner, L; Duronio, R J; Kishore, N S; Devadas, B; Adams, S P; Gordon, J I
1991-01-01
Covalent linkage of myristate (tetradecanoate; 14:0) to the NH2-terminal glycine residue of the human immunodeficiency virus 1 (HIV-1) 55-kDa gag polyprotein precursor (Pr55gag) is necessary for its proteolytic processing and viral assembly. We have shown recently that several analogs of myristate in which a methylene group is replaced by a single oxygen or sulfur atom are substrates for Saccharomyces cerevisiae and mammalian myristoyl-CoA:protein N-myristoyltransferase (EC 2.3.1.97; NMT) des...
Electron-capture ratios in radio-active decay
International Nuclear Information System (INIS)
Pelt, J. van.
1978-01-01
A review of the theory of electron capture is given and expressions for the electron-capture ratios derived. Attention is paid to the calculations of exchange and overlap correction factors. The theoretical results are then compared with experimental values on the basis of two recently published compilations. The experimental set-up used in this investigation is described and the analysis of the scintillation spectra discussed. Measurements on 131 Ba, 106 Agsup(m) and 196 Au are described and values derived for the exchange and overlap corrections and for the atomic electron-capture transition energy Qsub(EC). (Auth.)
International Nuclear Information System (INIS)
Pankratov, I. M.; Zhou, R. J.; Hu, L. Q.
2015-01-01
Peculiar phenomena were observed during experiments with runaway electrons: rapid changes in the synchrotron spot and its intensity that coincided with stepwise increases in the electron cyclotron emission (ECE) signal (cyclotron radiation of suprathermal electrons). These phenomena were initially observed in TEXTOR (Tokamak Experiment for Technology Oriented Research), where these events only occurred in the current decay phase or in discharges with thin stable runaway beams at a q = 1 drift surface. These rapid changes in the synchrotron spot were interpreted by the TEXTOR team as a fast pitch angle scattering event. Recently, similar rapid changes in the synchrotron spot and its intensity that coincided with stepwise increases in the non-thermal ECE signal were observed in the EAST (Experimental Advanced Superconducting Tokamak) runaway discharge. Runaway electrons were located around the q = 2 rational magnetic surface (ring-like runaway electron beam). During the EAST runaway discharge, stepwise ECE signal increases coincided with enhanced magnetohydrodynamic (MHD) activity. This behavior was peculiar to this shot. In this paper, we show that these non-thermal ECE step-like jumps were related to the abrupt growth of suprathermal electrons induced by bursting electric fields at reconnection events during this MHD plasma activity. Enhancement of the secondary runaway electron generation also occurred simultaneously. Local changes in the current-density gradient appeared because of local enhancement of the runaway electron generation process. These current-density gradient changes are considered to be a possible trigger for enhancement of the MHD plasma activity and the rapid changes in runaway beam behavior
Energy Technology Data Exchange (ETDEWEB)
Pankratov, I. M., E-mail: pankratov@kipt.kharkov.ua, E-mail: rjzhou@ipp.ac.cn [Institute of Plasma Physics, NSC Kharkov Institute of Physics and Technology, Academicheskaya Str. 1, 61108 Kharkov (Ukraine); Zhou, R. J., E-mail: pankratov@kipt.kharkov.ua, E-mail: rjzhou@ipp.ac.cn; Hu, L. Q. [Institute of Plasma Physics, Chinese Academy of Sciences, Hefei 230031 (China)
2015-07-15
Peculiar phenomena were observed during experiments with runaway electrons: rapid changes in the synchrotron spot and its intensity that coincided with stepwise increases in the electron cyclotron emission (ECE) signal (cyclotron radiation of suprathermal electrons). These phenomena were initially observed in TEXTOR (Tokamak Experiment for Technology Oriented Research), where these events only occurred in the current decay phase or in discharges with thin stable runaway beams at a q = 1 drift surface. These rapid changes in the synchrotron spot were interpreted by the TEXTOR team as a fast pitch angle scattering event. Recently, similar rapid changes in the synchrotron spot and its intensity that coincided with stepwise increases in the non-thermal ECE signal were observed in the EAST (Experimental Advanced Superconducting Tokamak) runaway discharge. Runaway electrons were located around the q = 2 rational magnetic surface (ring-like runaway electron beam). During the EAST runaway discharge, stepwise ECE signal increases coincided with enhanced magnetohydrodynamic (MHD) activity. This behavior was peculiar to this shot. In this paper, we show that these non-thermal ECE step-like jumps were related to the abrupt growth of suprathermal electrons induced by bursting electric fields at reconnection events during this MHD plasma activity. Enhancement of the secondary runaway electron generation also occurred simultaneously. Local changes in the current-density gradient appeared because of local enhancement of the runaway electron generation process. These current-density gradient changes are considered to be a possible trigger for enhancement of the MHD plasma activity and the rapid changes in runaway beam behavior.
Pankratov, I. M.; Zhou, R. J.; Hu, L. Q.
2015-07-01
Peculiar phenomena were observed during experiments with runaway electrons: rapid changes in the synchrotron spot and its intensity that coincided with stepwise increases in the electron cyclotron emission (ECE) signal (cyclotron radiation of suprathermal electrons). These phenomena were initially observed in TEXTOR (Tokamak Experiment for Technology Oriented Research), where these events only occurred in the current decay phase or in discharges with thin stable runaway beams at a q = 1 drift surface. These rapid changes in the synchrotron spot were interpreted by the TEXTOR team as a fast pitch angle scattering event. Recently, similar rapid changes in the synchrotron spot and its intensity that coincided with stepwise increases in the non-thermal ECE signal were observed in the EAST (Experimental Advanced Superconducting Tokamak) runaway discharge. Runaway electrons were located around the q = 2 rational magnetic surface (ring-like runaway electron beam). During the EAST runaway discharge, stepwise ECE signal increases coincided with enhanced magnetohydrodynamic (MHD) activity. This behavior was peculiar to this shot. In this paper, we show that these non-thermal ECE step-like jumps were related to the abrupt growth of suprathermal electrons induced by bursting electric fields at reconnection events during this MHD plasma activity. Enhancement of the secondary runaway electron generation also occurred simultaneously. Local changes in the current-density gradient appeared because of local enhancement of the runaway electron generation process. These current-density gradient changes are considered to be a possible trigger for enhancement of the MHD plasma activity and the rapid changes in runaway beam behavior.
Wang, Jianhao; Fan, Jie; Liu, Li; Ding, Shumin; Liu, Xiaoqian; Wang, Jianpeng; Gao, Liqian; Chattopadhaya, Souvik; Miao, Peng; Xia, Jiang; Qiu, Lin; Jiang, Pengju
2017-10-01
Herein, a novel assay has been developed for monitoring PreScission protease (His-PSP) mediated enzyme cleavage of ATTO 590 labeled peptide substrate (ATTO-LEV). This novel method is based on combining the use of capillary electrophoresis and fluorescence detection (CE-FL) to dynamically monitor the enzyme cleavage activity. A multivalent peptide substrate was first constructed by immobilizing His-tagged ATTO 590 labeled peptide substrate (ATTO-LEVH6) onto the surface of CdSe/ZnS quantum dots (QDs). Once successfully immobilized, the novel multivalent peptide substrate resulted in the Förster resonance energy transfer (FRET) from QDs to ATTO 590. The ATTO-LEVH6-QD assembly was then incubated with His-PSP to study the proteolytic cleavage of surface bound ATTO-LEVH6 by CE-FL. Our data suggests that PreScission-mediated proteolytic cleavage is enzyme concentration- and incubation time-dependent. By combining capillary electrophoresis, QDs and FRET, our study herein not only provides a new method for the detection and dynamically monitoring of PSP enzyme cleavage activity, but also can be extended to the detection of many other enzymes and proteases. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.
Dual functionality of β-tryptase protomers as both proteases and cofactors in the active tetramer.
Maun, Henry R; Liu, Peter S; Franke, Yvonne; Eigenbrot, Charles; Forrest, William F; Schwartz, Lawrence B; Lazarus, Robert A
2018-04-16
Human β-tryptase, a tetrameric trypsin-like serine protease, is an important mediator of the allergic inflammatory responses in asthma. During acute hypersensitivity reactions, mast cells degranulate, releasing active tetramer as a complex with proteoglycans. Extensive efforts have focused on developing therapeutic β-tryptase inhibitors, but its unique activation mechanism is less well explored. Tryptase is active only after proteolytic removal of the pro-domain followed by tetramer formation via two distinct symmetry-related interfaces. We show that the cleaved I16G mutant cannot tetramerize, likely due to impaired insertion of its N-terminus into its 'activation pocket', indicating allosteric linkage at multiple sites on each protomer. We engineered cysteines into each of the two distinct interfaces (Y75C for small or I99C for large) to assess the activity of each tetramer and disulfide-locked dimer. Using size-exclusion chromatography and enzymatic assays, we demonstrate that the two large tetramer interfaces regulate enzymatic activity, elucidating the importance of this protein-protein interaction for allosteric regulation. Notably, the I99C large interface dimer is active, even in the absence of heparin. We show that a monomeric β-tryptase mutant (I99C*:Y75A:Y37bA where C* is cysteinylated Cys99) cannot form a dimer or tetramer, yet is active, but only in the presence of heparin. Thus heparin both stabilizes the tetramer and allosterically conditions the active site. We hypothesize that each β-tryptase protomer in the tetramer has two distinct roles, acting both as a protease and as a cofactor for its neighboring protomer, to allosterically regulate enzymatic activity, providing a rationale for direct correlation of tetramer stability with proteolytic activity. Copyright © 2018, The American Society for Biochemistry and Molecular Biology.
Directory of Open Access Journals (Sweden)
Rezeda A. Akhmadishina
2018-02-01
Full Text Available Although delocalized lipophilic cations have been identified as effective cellular and mitochondrial carriers for a range of natural and synthetic drug molecules, little is known about their effects on pharmacological properties of peptides. The effect of triphenylphosphonium (TPP cation on bioactivity of antioxidant tetrapeptides based on the model opioid YRFK motif was studied. Two tetrapeptide variants with L-arginine (YRFK and D-arginine (YrFK were synthesized and coupled with carboxyethyl-TPP (TPP-3 and carboxypentyl-TPP (TPP-6 units. The TPP moiety noticeably promoted YRFK cleavage by trypsin, but effectively prevented digestion of more resistant YrFK attributed, respectively, to structure-organizing and shielding effects of the TPP cation on conformational variants of the tetrapeptide motif. The TPP moiety enhanced radical scavenging activity of the modified YRFK in a model Fenton-like reaction, whereas decreased reactivity was revealed for both YrFK and its TPP derivative. The starting motifs and modified oligopeptides, especially the TPP-6 derivatives, suppressed acute oxidative stress in neuronal PC-12 cells during a brief exposure similarly with glutathione. The effect of oligopeptides was compared upon culturing of PC-12 cells with CoCl2, L-glutamic acid, or menadione to mimic physiologically relevant oxidative states. The cytoprotective activity of oligopeptides significantly depended on the type of oxidative factor, order of treatment and peptide structure. Pronounced cell-protective effect was established for the TPP-modified oligopeptides, which surpassed that of the unmodified motifs. The protease-resistant TPP-modified YrFK showed the highest activity when administered 24 h prior to the cell damage. Our results suggest that the TPP cation can be used as a modifier for small therapeutic peptides to improve their pharmacokinetic and pharmacological properties.
Practical electronics handbook
Sinclair, Ian R
2013-01-01
Practical Electronics Handbook, Third Edition provides the frequently used and highly applicable principles of electronics and electronic circuits.The book contains relevant information in electronics. The topics discussed in the text include passive and active discrete components; linear and digital I.C.s; microprocessors and microprocessor systems; digital-analogue conversions; computer aids in electronics design; and electronic hardware components.Electronic circuit constructors, service engineers, electronic design engineers, and anyone with an interest in electronics will find the book ve
DEFF Research Database (Denmark)
Nielsen, Maria Ravn; Rytz, Mie; Frederiksen-Møller, Britta
2015-01-01
OBJECTIVES: Increased activity of the epithelial sodium channel (ENaC) in the kidneys may explain the coupling between proteinuria, edema, suppressed aldosterone and hypertension in preeclampsia. Preeclamptic women excrete plasminogen-plasmin in urine. In vitro, plasmin increases the activity...... as a positive control for the presence of collecting duct membrane. RESULTS: Urine plasmin-plasminogen/creatinine ratio was increased in the preeclampsia group (p... pregnancy and preeclampsia CONCLUSIONS: It is possible to examine collecting duct transport proteins in urine exosome from pregnant women including γ-ENaC, 2) Urine exosome fraction displays a variable pattern of γ-ENaC signal with a predominance of cleaved forms in both normal and preeclamptic women...
International Nuclear Information System (INIS)
Gun’ko, V.M.; Zaulychnyy, Ya.V.; Ilkiv, B.I.; Zarko, V.I.; Nychiporuk, Yu.M.; Pakhlov, E.M.; Ptushinskii, Yu.G.; Leboda, R.; Skubiszewska-Zięba, J.
2011-01-01
Nanosilicas (A-50, A-300, A-500)/activated carbon (AC, S BET = 1520 m 2 /g) composites were prepared using short-term (5 min) mechanochemical activation (MCA) of powder mixtures in a microbreaker. Smaller silica nanoparticles of A-500 (average diameter d av = 5.5 nm) can more easily penetrate into broad mesopores and macropores of AC microparticles than larger nanoparticles of A-50 (d av = 52.4 nm) or A-300 (d av = 8.1 nm). After MCA of silica/AC, nanopores of non-broken AC nanoparticles remained accessible for adsorbed N 2 molecules. According to ultra-soft X-ray emission spectra (USXES), MCA of silica/AC caused formation of chemical bonds Si-O-C; however, Si-C and Si-Si bonds were practically not formed. A decrease in intensity of OK α band in respect to CK α band of silica/AC composites with diminishing sizes of silica nanoparticles is due to both changes in the surface structure of particles and penetration of a greater number of silica nanoparticles into broad pores of AC microparticles and restriction of penetration depth of exciting electron beam into the AC particles.
Multitarget Molecular Hybrids of Cinnamic Acids
Directory of Open Access Journals (Sweden)
Aikaterini Peperidou
2014-12-01
Full Text Available In an attempt to synthesize potential new multitarget agents, 11 novel hybrids incorporating cinnamic acids and paracetamol, 4-/7-hydroxycoumarin, benzocaine, p-aminophenol and m-aminophenol were synthesized. Three hybrids—2e, 2a, 2g—and 3b were found to be multifunctional agents. The hybrid 2e derived from the phenoxyphenyl cinnamic acid and m-acetamidophenol showed the highest lipoxygenase (LOX inhibition and analgesic activity (IC50 = 0.34 μΜ and 98.1%, whereas the hybrid 3b of bromobenzyloxycinnamic acid and hymechromone exhibited simultaneously good LOX inhibitory activity (IC50 = 50 μΜ and the highest anti-proteolytic activity (IC50= 5 μΜ. The hybrid 2a of phenyloxyphenyl acid with paracetamol showed a high analgesic activity (91% and appears to be a promising agent for treating peripheral nerve injuries. Hybrid 2g which has an ester and an amide bond presents an interesting combination of anti-LOX and anti-proteolytic activity. The esters were found very potent and especially those derived from paracetamol and m-acetamidophenol. The amides follow. Based on 2D-structure–activity relationships it was observed that both steric and electronic parameters play major roles in the activity of these compounds. Molecular docking studies point to the fact that allosteric interactions might govern the LOX-inhibitor binding.
Tonaco, Luís A B; Gomes, Flavia L; Velasquez-Melendez, Gustavo; Lopes, Miriam T P; Salas, Carlos E
2018-04-01
The aim of the study was to investigate the role of the proteolytic fraction from Vasconcellea cundinamarcensis, designated as P1G10, on the healing of chronic foot ulcers in neuropathic patients with diabetes 2. Fifty patients were enrolled in a prospective, randomized, double-blind trial, to verify the efficacy and safety of a topical dressing formulated with 0.1% P1G10, intended for wound healing, versus a hydrogel (control) protocol. Upon completion of the intervention, the outcome evaluated the number of patients attaining full epithelization (100%), or at least 80% healing. Statistical analysis compared the data on each group for the significance of the differences. Collection of data was finished in week 16, and the results were analyzed by intention to treat. The results showed that, in the control group, 5 patients attained 100% ulcer healing, 3 patients ≥ 80% healing and 11 experienced ulcer changes ≤ 80%, and the remainder showed no changes or their wounds became worse. Meanwhile, in the P1G10 group, 11 patients experienced full healing, 4 had healing ≥ 80% and 5 had ulcer changes ≤ lower than 80%, and the remainder showed no changes or their wounds became worse. The healing incidence for the first endpoint (100% healing) showed that the P1G10 group was 2.95-fold more efficacious than the control group (CI 95%) and 2.52-fold (CI, 95%) higher than its control for the second endpoint (80% healing). These data support the hypothesis that topical application of the proteolytic fraction identified as P1G10 significantly enhances foot ulcer healing compared to hydrogel treatment.
Photonic activation of plasminogen induced by low dose UVB.
Directory of Open Access Journals (Sweden)
Manuel Correia
Full Text Available Activation of plasminogen to its active form plasmin is essential for several key mechanisms, including the dissolution of blood clots. Activation occurs naturally via enzymatic proteolysis. We report that activation can be achieved with 280 nm light. A 2.6 fold increase in proteolytic activity was observed after 10 min illumination of human plasminogen. Irradiance levels used are in the same order of magnitude of the UVB solar irradiance. Activation is correlated with light induced disruption of disulphide bridges upon UVB excitation of the aromatic residues and with the formation of photochemical products, e.g. dityrosine and N-formylkynurenine. Most of the protein fold is maintained after 10 min illumination since no major changes are observed in the near-UV CD spectrum. Far-UV CD shows loss of secondary structure after illumination (33.4% signal loss at 206 nm. Thermal unfolding CD studies show that plasminogen retains a native like cooperative transition at ~70 ºC after UV-illumination. We propose that UVB activation of plasminogen occurs upon photo-cleavage of a functional allosteric disulphide bond, Cys737-Cys765, located in the catalytic domain and in van der Waals contact with Trp761 (4.3 Å. Such proximity makes its disruption very likely, which may occur upon electron transfer from excited Trp761. Reduction of Cys737-Cys765 will result in likely conformational changes in the catalytic site. Molecular dynamics simulations reveal that reduction of Cys737-Cys765 in plasminogen leads to an increase of the fluctuations of loop 760-765, the S1-entrance frame located close to the active site. These fluctuations affect the range of solvent exposure of the catalytic triad, particularly of Asp646 and Ser74, which acquire an exposure profile similar to the values in plasmin. The presented photonic mechanism of plasminogen activation has the potential to be used in clinical applications, possibly together with other enzymatic treatments for the
Directory of Open Access Journals (Sweden)
Guowei Shu
2018-05-01
Full Text Available Despite the widespread application of lactic acid bacterium in dairy production through its contribution to acidification, development of sensorial properties, and health-promoting effects, relatively little information is available on the cell envelope proteinases (CEPs of Lactobacillus plantarum, especially on the proteolytic system and the production of bioactivity peptides. In this study, CEPs from a novel L. plantarum LP69 were involved in goat milk hydrolysis and generated a product with high activity that showed a degree of hydrolysis of 15.68 ± 0.74%, Angiotensin I-Converting Enzyme (ACE-inhibitory rate of 83.25 ± 1.05%, 2,2-diphenyl-1-picrylhydrazyl (DPPH radical scavenging rate of 64.91 ± 1.27%, and hydroxyl radical scavenging rate of 89.17 ± 1.13%. The optimized hydrolysis conditions were time of 4.5 h, temperature of 41 °C, initial pH of 8.5, and enzyme to substrate ratio (E/S of 12% (w/w by orthogonal experiments. Application of a stabilizer greatly promoted milk stability. A well-designed stabilizer consists of 0.05% carrageenan, 0.15% gellan gum, and 0.15% sucrose esters, which significantly raised the milk stability coefficient, R, from 70.67% to 98.57%. The storage stability of milk was evaluated during 84 days at room temperature or 4 °C. Our study depicts the contribution of CEPs from L. plantarum LP69 in goat milk, exploring a new way for the development of a functional milk product.
International Nuclear Information System (INIS)
Blanc-Brude, Olivier P.; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.
2005-01-01
Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR 1 ). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR 1 -deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR 1 -specific agonists and inhibitors were used to demonstrate that PAR 1 mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR 1 and not PAR 2 . These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis
Active Pixel Sensors for electron microscopy
Energy Technology Data Exchange (ETDEWEB)
Denes, P. [Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)], E-mail: pdenes@lbl.gov; Bussat, J.-M. [Engineering Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States); Lee, Z.; Radmillovic, V. [National Center for Electron Microscopy, Lawrence Berkeley National Laboratory, Berkeley, CA 94720 (United States)
2007-09-01
The technology used for monolithic CMOS imagers, popular for cell phone cameras and other photographic applications, has been explored for charged particle tracking by the high-energy physics community for several years. This technology also lends itself to certain imaging detector applications in electron microscopy. We have been developing such detectors for several years at Lawrence Berkeley National Laboratory, and we and others have shown that this technology can offer excellent point-spread function, direct detection and high readout speed. In this paper, we describe some of the design constraints peculiar to electron microscopy and summarize where such detectors could play a useful role.
International Nuclear Information System (INIS)
Ji, Xiaoli; Xu, Bo; Yao, Mengmeng; Mao, Zhilei; Zhang, Yuqing; Xu, Guofeng; Tang, Qiusha; Wang, Xinru; Xia, Yankai
2016-01-01
Graphene oxide quantum dots (GOQDs) have broad application prospects in many areas including bioimaging, drug delivery, DNA cleavage system, sensors and photocatalyst. Recently, increasing concerns have been raised about their biocompatibility, but studies about the effects of GOQDs on male reproductive system are still lacking. In this work, we explored the effects and molecular mechanisms of GOQDs on GC-2 and TM4 cells. We found autophagosome accumulation in GC-2 and TM4 cells after GOQDs treatment. Both LC3-II/LC3-I ratio and p62 levels increased, and the chloroquine-induced accumulation of LC3-II didn’t enhance in the presence of GOQDs, which indicated that GOQDs blocked autophagic flux. Further studies found that the fusion between autophagosome and lysosome was not inhibited by GOQDs, but the proteolytic capacity of lysosome was weakened and both the expression and activity of cathepsin B reduced. Taken together, these results suggested that GOQDs blocked autophagic flux by decreasing the amount and enzymatic activity of cathepsin B and inhibiting lysosome proteolytic capacity in GC-2 and TM4 cells, which might have a potential hazard to male reproduction.
Directory of Open Access Journals (Sweden)
M. Budič
2008-09-01
Full Text Available A wide variety of proteolytic enzymes exist in plants. On their levels depends protein turnover, a fundamental component in plant development and adaptation to environmental conditions. Cysteine proteases have frequently been reported to be influenced by drought, but only a few serine proteases (SP, among them the trypsin-like enzyme and two aminopeptidases from bean leaves (Bartels and Sunkar, 2005; Hieng et al., 2004. Our starting point was to identify proteolytic activities assigned to SPs that change with drought and then to characterize the corresponding proteases. A quantitative, analytical one-step method was used to separate endopeptidases and aminopeptidases active against a range of substrates in leaf extracts of plants grown in the field (FC. The influence of drought was determined for those of these activities which were confirmed as SPs, based on their inhibition by specific inhibitors. Under water deficit in plants grown under controlled conditions (CC their levels changed in different ways. The levels of SP activities in FC plants, observed during a period of relative drought, were similar to those measured in mildly stressed CC plants. The partial characterisations of some of these SPs will be presented. Our results point to a number of roles for different SPs in the plant response to water stress, which could range from enhanced protein turnover to limited proteolysis at specific sites.
Latitudinal variation of the topside electron temperature at different levels of solar activity
Czech Academy of Sciences Publication Activity Database
Truhlík, Vladimír; Bilitza, D.; Třísková, Ludmila
2009-01-01
Roč. 44, č. 6 (2009), s. 693-700 ISSN 0273-1177 R&D Projects: GA AV ČR IAA300420603 Grant - others: NASA (US) NNH06CD17C Institutional research plan: CEZ:AV0Z30420517 Keywords : Electron temperature * Solar activity variation * Latitudinal dependence Subject RIV: DG - Athmosphere Sciences, Meteorology Impact factor: 1.079, year: 2009
Xie, Xiujun; Gu, Wenhui; Gao, Shan; Lu, Shan; Li, Jian; Pan, Guanghua; Wang, Guangce; Shen, Songdong
2013-01-01
The xanthophyll cycle (Xc), which involves violaxanthin de-epoxidase (VDE) and the zeaxanthin epoxidase (ZEP), is one of the most rapid and efficient responses of plant and algae to high irradiance. High light intensity can activate VDE to convert violaxanthin (Vx) to zeaxanthin (Zx) via antheraxanthin (Ax). However, it remains unclear whether VDE remains active under low light or dark conditions when there is no significant accumulation of Ax and Zx, and if so, how the ΔpH required for activation of VDE is built. In this study, we used salicylaldoxime (SA) to inhibit ZEP activity in the intertidal macro-algae Ulva sp. (Ulvales, Chlorophyta) and then characterized VDE under low light and dark conditions with various metabolic inhibitors. With inhibition of ZEP by SA, VDE remained active under low light and dark conditions, as indicated by large accumulations of Ax and Zx at the expense of Vx. When PSII-mediated linear electron transport systems were completely inhibited by SA and DCMU, alternative electron transport systems (i.e., cyclic electron transport and chlororespiration) could maintain VDE activity. Furthermore, accumulations of Ax and Zx decreased significantly when SA, DCMU, or DBMIB together with an inhibitor of chlororespiration (i.e., propyl gallate (PG)) were applied to Ulva sp. This result suggests that chlororespiration not only participates in the build-up of the necessary ΔpH, but that it also possibly influences VDE activity indirectly by diminishing the oxygen level in the chloroplast.
Directory of Open Access Journals (Sweden)
Xiujun Xie
Full Text Available The xanthophyll cycle (Xc, which involves violaxanthin de-epoxidase (VDE and the zeaxanthin epoxidase (ZEP, is one of the most rapid and efficient responses of plant and algae to high irradiance. High light intensity can activate VDE to convert violaxanthin (Vx to zeaxanthin (Zx via antheraxanthin (Ax. However, it remains unclear whether VDE remains active under low light or dark conditions when there is no significant accumulation of Ax and Zx, and if so, how the ΔpH required for activation of VDE is built. In this study, we used salicylaldoxime (SA to inhibit ZEP activity in the intertidal macro-algae Ulva sp. (Ulvales, Chlorophyta and then characterized VDE under low light and dark conditions with various metabolic inhibitors. With inhibition of ZEP by SA, VDE remained active under low light and dark conditions, as indicated by large accumulations of Ax and Zx at the expense of Vx. When PSII-mediated linear electron transport systems were completely inhibited by SA and DCMU, alternative electron transport systems (i.e., cyclic electron transport and chlororespiration could maintain VDE activity. Furthermore, accumulations of Ax and Zx decreased significantly when SA, DCMU, or DBMIB together with an inhibitor of chlororespiration (i.e., propyl gallate (PG were applied to Ulva sp. This result suggests that chlororespiration not only participates in the build-up of the necessary ΔpH, but that it also possibly influences VDE activity indirectly by diminishing the oxygen level in the chloroplast.
Are associations between electronic media use and BMI different across levels of physical activity?
DEFF Research Database (Denmark)
Melkevik, Ole; Haug, Ellen; Rasmussen, Mette
2015-01-01
and girls who did not comply with physical activity guidelines. Among physically active adolescents, EM was found to be significantly associated with BMI or odds for overweight among girls, but not among boys. CONCLUSION: While the usage of EM appear to be inconsequential for BMI and the risk of overweight...... among physically active boys, we find evidence indicating that EM use is associated with BMI and risk for overweight among girls, including those who report complying with MVPA guidelines.......BACKGROUND: The use of electronic media has been found to be a risk factor for higher BMI and for being overweight. Physical activity has been found to be associated with lower BMI and lower risk for being overweight. Little is known about whether the associations between physical activity...
International Nuclear Information System (INIS)
Poccia, N.; Ricci, A.; Bianconi, N.
2010-01-01
High-temperature superconductivity (HTS) emerges in quite different electronic materials: cuprates, diborides, and iron-pnictide superconductors. Looking for unity in the diversity we find in all these materials a common lattice architecture: they are practical realizations of heterostructures at atomic limit made of superlattices of metallic active layers intercalated by spacers as predicted in 1993 by one of us. The multilayer architecture is the key feature for the presence of electronic topological transitions where the Fermi surface of one of the subbands changes dimensionality. The superlattice misfit strain η between the active and spacer layers is shown to be a key variable to drive the system to the highest critical temperature Tc that occurs at a particular point of the 3D phase diagram Tc(θ, η) where d is the charge transfer or doping. The plots of Tc as a function of misfit strain at constant charge transfer in cuprates show a first-order quantum critical phase transition where an itinerant striped magnetic phase competes with superconductivity in the proximity of a structural phase transition, that is, associated with an electronic topological transition. The shape resonances in these multi gap superconductors is associated with the maximum Tc.
Bothrops fonsecai snake venom activities and cross-reactivity with commercial bothropic venom.
Collaço, Rita de Cássia O; Randazzo-Moura, Priscila; Tamascia, Mariana L; da Silva, Igor Rapp F; Rocha, Thalita; Cogo, José C; Hyslop, Stephen; Sanny, Charles G; Rodrigues-Simioni, Léa
2017-01-01
In this work, we examined some biochemical and biological activities of Bothrops fonsecai venom, a pitviper endemic to southeastern Brazil, and assessed their neutralization by commercial bothropic antivenom (CAv). Cross-reactivity of venom with CAv was also assessed by immunoblotting and size-exclusion high performance chromatography (SE-HPLC). Bothrops fonsecai venom had PLA 2 , proteolytic and esterase activities that were neutralized to varying extents by venom:antivenom ratios of 5:1 and 5:2 (PLA 2 and esterase activities) or not significantly by either venom:antivenom ratio (proteolytic activity). The minimum hemorrhagic dose (69.2μg) was totally neutralized by both ratios. Clotting time in rat citrated plasma was 33±10.5s (mean±SD; n=5) and was completely neutralized by a 5:2 ratio. Edema formation was dose-dependent (1-30μg/site) and significantly inhibited by both ratios. Venom (10-300μg/mL) caused neuromuscular blockade in extensor digitorum longus preparations; this blockade was inhibited best by a 5:2 ratio. Venom caused myonecrosis and creatine kinase release in vivo (gastrocnemius muscle) and in vitro (extensor digitorum longus) that was effectively neutralized by both venom:antivenom ratios. Immunoblotting showed that venom components of ~25-100kDa interacted with CAv. SE-HPLC profiles for venom incubated with CAv or specific anti-B. fonsecai antivenom raised in rabbits (SAv) indicated that CAv had a higher binding capacity than SAv, whereas SAv had higher affinity than CAv. These findings indicate that B. fonsecai venom contains various activities that are neutralized to different extents by CAv and suggest that CAv could be used to treat envenoming by B. fonsecai. Copyright © 2016. Published by Elsevier Inc.
Directory of Open Access Journals (Sweden)
Anna Prudova
2016-08-01
Full Text Available Deregulated cathepsin proteolysis occurs across numerous cancers, but in vivo substrates mediating tumorigenesis remain ill-defined. Applying 8-plex iTRAQ terminal amine isotopic labeling of substrates (TAILS, a systems-level N-terminome degradomics approach, we identified cathepsin B, H, L, S, and Z in vivo substrates and cleavage sites with the use of six different cathepsin knockout genotypes in the Rip1-Tag2 mouse model of pancreatic neuroendocrine tumorigenesis. Among 1,935 proteins and 1,114 N termini identified by TAILS, stable proteolytic products were identified in wild-type tumors compared with one or more different cathepsin knockouts (17%–44% of 139 cleavages. This suggests a lack of compensation at the substrate level by other cathepsins. The majority of neo-N termini (56%–83% for all cathepsins was consistent with protein degradation. We validated substrates, including the glycolytic enzyme pyruvate kinase M2 associated with the Warburg effect, the ER chaperone GRP78, and the oncoprotein prothymosin-alpha. Thus, the identification of cathepsin substrates in tumorigenesis improves the understanding of cathepsin functions in normal physiology and cancer.
Energy Technology Data Exchange (ETDEWEB)
Ramakrishnan, Raghunathan, E-mail: r.ramakrishnan@unibas.ch [Institute of Physical Chemistry, National Center for Computational Design and Discovery of Novel Materials (MARVEL), Department of Chemistry, University of Basel, Klingelbergstrasse 80, CH-4056 Basel (Switzerland); Nest, Mathias [Theoretische Chemie, Technische Universität München, Lichtenbergstr. 4, 85747 Garching (Germany)
2015-01-13
Highlights: • We model electron dynamics across cyano alkanethiolates attached to gold cluster. • We present electron transfer time scales from TD-DFT and TD-CI based simulations. • Both DFT and CI methods qualitatively predict the trend in time scales. • TD-CI predicts the experimental relative time scale very accurately. - Abstract: We employ wavepacket simulations based on many-body time-dependent configuration interaction (TD-CI), and single active electron theories, to predict the ultrafast molecule/metal electron transfer time scales, in cyano alkanethiolates bonded to model gold clusters. The initial states represent two excited states where a valence electron is promoted to one of the two virtual π{sup ∗} molecular orbitals localized on the cyanide fragment. The ratio of the two time scales indicate the efficiency of one charge transfer channel over the other. In both our one-and many-electron simulations, this ratio agree qualitatively with each other as well as with the previously reported experimental time scales (Blobner et al., 2012), measured for a macroscopic metal surface. We study the effect of cluster size and the description of electron correlation on the charge transfer process.
International Nuclear Information System (INIS)
Sari, A.; Carrel, F.; Gmar, M.; Laine, F.; Normand, S.; Lyoussi, A.
2012-01-01
The solution for management of a nuclear waste package is chosen according to its radiological characteristics. One of the most important of these features is the α-activity which is due to actinides ( 235 U, 238 U, 239 Pu, etc.) If non-destructive passive methods are not sufficient to quantify the latter, non-destructive active methods based on the fission process represent a solution of interest. First, these methods consist in irradiating a package in order to induce fission reactions on the actinides, and then, to detect the prompt and delayed particles which are emitted following these reactions. Our aim is to conduct neutron interrogation measurements on nuclear waste packages using an electron accelerator as a photoneutron generator. One of the main interests of this approach is that the intensity of the neutron flux can be one or two orders of magnitude higher than the one delivered by a deuterium-tritium generator. With the objective of improving nuclear waste characterization, the development of this method could enable the integration of three complementary techniques on a single measurement cell (active neutron interrogation, active photon interrogation, and high-energy imaging). In this paper, simulation and experimental results are presented. A simulation study using MCNPX has been conducted in order to determine the characteristics of the photoneutron flux emitted by the electron accelerator of the SAPHIR facility owned by CEA LIST. Energy spectra, angular distribution and intensity of the photoneutron flux have been obtained. A photoneutron interrogation measurement cell based on this accelerator has been built and assessed by carrying out measurements on uranium samples. Delayed gamma-ray spectra have been acquired and enabled to confirm the experimental feasibility of our method. (authors)
Yucel, Nihal; Aslim, Belma; Ozdoğan, Hakan
2009-08-01
In this study a total of 30 raw meat samples obtained from Ankara, Turkey were screened for the presence of Bacillus species. Among the meat samples analyzed, the predominant species isolated was Bacillus circulans; other Bacillus species were identified as Bacillus firmus, Bacillus lentus, Bacillus megaterium, Bacillus licheniformis, Bacillus mycoides, Bacillus sphaericus, and Bacillus cereus. Minced meat samples were more contaminated with Bacillus species than sliced beef sample. From these samples, 242 Bacillus species isolates were obtained, which were investigated for proteolytic and lipolytic activity, associated with meat spoilage. Interestingly, some Bacillus strains produced the highest values of proteolytic/lipolytic activities. Nineteen Bacillus strains were selected among the 242 isolates according to their proteolytic/lipolytic activity with a clear zone diameter of > or =6 mm. The essential oil of Satureja wiedemanniana (Lalem) Velen was also tested against these 19 Bacillus species that had proteolytic and lipolytic activity. The essential oil yield obtained from the aerial parts of the plant was 0.35% (vol/wt). The inhibition zones of the essential oil obtained against all the Bacillus species were in the range of 5.0-12.0 mm. The oil showed high antimicrobial activities against B. licheniformis M 6(26), M 11(16), and M 12(1) strains. B. licheniformis 12(1) showed high lipolytic activity (18.0 mm). Also, B. licheniformis M 6(26) and M 11(16) showed high proteolytic activity (16.0 and 14.0 mm). These results may suggest that an essential oil of S. wiedemanniana can be used as a natural preservative in meat against spoilage bacteria.
Lopez-Bote, C J; Toldrá, F; Daza, A; Ferrer, J M; Menoyo, D; Silió, L; Rodríguez, M C
2008-05-01
The effects of physical activity on performance, carcass traits, Psoas major lysosomal and exoprotease acitivies and meat quality were studied in 24 castrated male Iberian pigs during the last fattening period (from 111.1±SD: 5.2kg). Pigs were randomly distributed in three groups. Two groups receiving the same diet were reared in confinement, one housed in individual pens of 8m(2) (sedentary group) and the other was housed outdoor with daily (up to 2km) forced walking (exercise group). And one group was reared under the traditional production system walking daily several km and fed mostly with acorn from Quercus ilex and Quercus rotundifolia and grass (free-range group). No differences were found in performance and carcass traits. In exercised pigs a lower activity of cathepsin B+L and total cathepsins (P<0.05) was observed. Exercise induced the inhibition of dipeptidyl peptidases II and III and arginyl aminopeptidase and the activation of dipeptidyl peptidases IV and leucyl aminopeptidase (P<0.05). Although no effects on total free amino acids in Psoas major muscle were observed the concentration of branched chain amino acids decreased in the free-range pig group probably related to an increase in physical activity. Exercise had no effects in Psoas major postmortem tenderness and water holding capacity.
Rueda, Analiz; Sifuentes, Cecilia; Gilman, Robert H; Gutiérrez, Andrés H; Piña, Ruby; Chile, Nancy; Carrasco, Sebastián; Larson, Sandra; Mayta, Holger; Verástegui, Manuela; Rodriguez, Silvia; Gutiérrez-Correa, Marcel; García, Héctor H; Sheen, Patricia; Zimic, Mirko
2011-12-01
Neurocysticercosis is an endemic parasitic disease caused by Taenia solium larva. Although the mechanism of infection is not completely understood, it is likely driven by proteolytic activity that degrades the intestinal wall to facilitate oncosphere penetration and further infection. We analyzed the publicly available T. solium EST/DNA library and identified two contigs comprising a full-length cDNA fragment very similar to Echinococcus granulosus Ag5 protein. The T. solium cDNA sequence included a proteolytic trypsin-like-domain in the C-terminal region, and a thrombospondin type-1 adherence-domain in the N-terminal region. Both the trypsin-like and adherence domains were expressed independently as recombinant proteins in bacterial systems. TsAg5 showed marginal trypsin-like activity and high sequence similarity to Ag5. The purified antigens were tested in a Western immunoblot assay to diagnose human neurocysticercosis. The sensitivity of the trypsin-like-domain was 96.36% in patients infected with extraparenchymal cysts, 75.44% in patients infected with multiple cysts, and 39.62% in patients with a single cyst. Specificity was 76.70%. The thrombospondin type-1 adherence-domain was not specific for neurocysticercosis. Copyright © 2011 Elsevier B.V. All rights reserved.
Nuclear electronics laboratory manual
International Nuclear Information System (INIS)
1984-05-01
The Nuclear Electronics Laboratory Manual is a joint product of several electronics experts who have been associated with IAEA activity in this field for many years. The manual does not include experiments of a basic nature, such as characteristics of different active electronics components. It starts by introducing small electronics blocks, employing one or more active components. The most demanding exercises instruct a student in the design and construction of complete circuits, as used in commercial nuclear instruments. It is expected that a student who completes all the experiments in the manual should be in a position to design nuclear electronics units and also to understand the functions of advanced commercial instruments which need to be repaired or maintained. The future tasks of nuclear electronics engineers will be increasingly oriented towards designing and building the interfaces between a nuclear experiment and a computer. The manual pays tribute to this development by introducing a number of experiments which illustrate the principles and the technology of interfacing
DEFF Research Database (Denmark)
Farver, O; Pecht, I
1991-01-01
Electron migration between and within proteins is one of the most prevalent forms of biological energy conversion processes. Electron transfer reactions take place between active centers such as transition metal ions or organic cofactors over considerable distances at fast rates and with remarkable...... specificity. The electron transfer is attained through weak electronic interaction between the active sites, so that considerable research efforts are centered on resolving the factors that control the rates of long-distance electron transfer reactions in proteins. These factors include (in addition......-containing proteins. These proteins serve almost exclusively in electron transfer reactions, and as it turns out, their metal coordination sites are endowed with properties uniquely optimized for their function....
Solari, Fiorella A; Mattheij, Nadine J A; Burkhart, Julia M; Swieringa, Frauke; Collins, Peter W; Cosemans, Judith M E M; Sickmann, Albert; Heemskerk, Johan W M; Zahedi, René P
2016-10-01
The Scott syndrome is a very rare and likely underdiagnosed bleeding disorder associated with mutations in the gene encoding anoctamin-6. Platelets from Scott patients are impaired in various Ca 2+ -dependent responses, including phosphatidylserine exposure, integrin closure, intracellular protein cleavage, and cytoskeleton-dependent morphological changes. Given the central role of anoctamin-6 in the platelet procoagulant response, we used quantitative proteomics to understand the underlying molecular mechanisms and the complex phenotypic changes in Scott platelets compared with control platelets. Therefore, we applied an iTRAQ-based multi-pronged strategy to quantify changes in (1) the global proteome, (2) the phosphoproteome, and (3) proteolytic events between resting and stimulated Scott and control platelets. Our data indicate a limited number of proteins with decreased (70) or increased (64) expression in Scott platelets, among those we confirmed the absence of anoctamin-6 and the strong up-regulation of aquaporin-1 by parallel reaction monitoring. The quantification of 1566 phosphopeptides revealed major differences between Scott and control platelets after stimulation with thrombin/convulxin or ionomycin. In Scott platelets, phosphorylation levels of proteins regulating cytoskeletal or signaling events were increased. Finally, we quantified 1596 N-terminal peptides in activated Scott and control platelets, 180 of which we identified as calpain-regulated, whereas a distinct set of 23 neo-N termini was caspase-regulated. In Scott platelets, calpain-induced cleavage of cytoskeleton-linked and signaling proteins was downregulated, in accordance with an increased phosphorylation state. Thus, multipronged proteomic profiling of Scott platelets provides detailed insight into their protection against detrimental Ca 2+ -dependent changes that are normally associated with phosphatidylserine exposure. © 2016 by The American Society for Biochemistry and Molecular
Pshenichnyuk, Stanislav A; Elkin, Yury N; Kulesh, Nadezda I; Lazneva, Eleonora F; Komolov, Alexei S
2015-07-14
The antioxidant isoflavone retusin efficiently attaches low-energy electrons in vacuo, generating fragment species via dissociative electron attachment (DEA), as has been shown by DEA spectroscopy. According to in silico results obtained by means of density functional theory, retusin is able to attach solvated electrons and could be decomposed under reductive conditions in vivo, for instance, near the mitochondrial electron transport chain, analogous to gas-phase DEA. The most intense decay channels of retusin temporary negative ions were found to be associated with the elimination of H atoms and H2 molecules. Doubly dehydrogenated fragment anions were predicted to possess a quinone structure. It is thought that molecular hydrogen, known for its selective antioxidant properties, can be efficiently generated via electron attachment to retusin in mitochondria and may be responsible for its antioxidant activity. The second abundant species, i.e., quinone bearing an excess negative charge, can serve as an electron carrier and can return the captured electron back to the respiration cycle. The number of OH substituents and their relative positions are crucial for the present molecular mechanism, which can explain the radical scavenging activity of polyphenolic compounds.
Rapid tryptic mapping using enzymatically active mass spectrometer probe tips
Energy Technology Data Exchange (ETDEWEB)
Dogruel, D.; Williams, P.; Nelson, R.W. [Arizona State Univ., Tempe, AZ (United States)
1995-12-01
A method has been developed for rapid, sensitive, and accurate tryptic mapping of polypeptides using matrix-assisted laser desorption/ionization time-of-flight mass analysis. The technique utilizes mass spectrometer probe tips which have been activated through the covalent immobilization of trypsin. The enzymatically active probe tips were used for the tryptic mapping of chicken egg lysozyme and the results compared with those obtained using either free trypsin or agarose-immobilized trypsin. A significant increase in the overall sensitivity of the process was observed using the active probe tips, as well as the production of more characteristic proteolytic fragments and the elimination of background signals due to the autolysis of the trypsin. Further, probe tip digestions were found to be rapid and convenient. 19 refs., 6 figs., 2 tabs.
Antibody proteases: induction of catalytic response.
Gabibov, A G; Friboulet, A; Thomas, D; Demin, A V; Ponomarenko, N A; Vorobiev, I I; Pillet, D; Paon, M; Alexandrova, E S; Telegin, G B; Reshetnyak, A V; Grigorieva, O V; Gnuchev, N V; Malishkin, K A; Genkin, D D
2002-10-01
Most of the data accumulated throughout the years on investigation of catalytic antibodies indicate that their production increases on the background of autoimmune abnormalities. The different approaches to induction of catalytic response toward recombinant gp120 HIV-1 surface protein in mice with various autoimmune pathologies are described. The peptidylphosphonate conjugate containing structural part of gp120 molecule is used for reactive immunization of NZB/NZW F1, MRL, and SJL mice. The specific modification of heavy and light chains of mouse autoantibodies with Val-Ala-Glu-Glu-Glu-Val-PO(OPh)2 reactive peptide was demonstrated. Increased proteolytic activity of polyclonal antibodies in SJL mice encouraged us to investigate the production of antigen-specific catalytic antibodies on the background of induced experimental autoimmune encephalomyelitis (EAE). The immunization of autoimmune-prone mice with the engineered fusions containing the fragments of gp120 and encephalitogenic epitope of myelin basic protein (MBP(89-104)) was made. The proteolytic activity of polyclonal antibodies isolated from the sera of autoimmune mice immunized by the described antigen was shown. Specific immune response of SJL mice to these antigens was characterized. Polyclonal antibodies purified from sera of the immunized animals revealed proteolytic activity. The antiidiotypic approach to raise the specific proteolytic antibody as an "internal image" of protease is described. The "second order" monoclonal antibodies toward subtilisin Carlsberg revealed pronounced proteolytic activity.
Characterization of active-site residues of the NIa protease from tobacco vein mottling virus.
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.
Activity of Lactobacillus casei and its gamma-radiation induced mutant in different types of milk
International Nuclear Information System (INIS)
Singh, J.; Ranganathan, B.
1979-01-01
Lactobacillus casei (RTS) and one of its gamma-radiation induced mutants, selected on the basis of increased proteolytic activity were individually inoculated in skimmed milk samples of different species. After incubation at 37 0 C for 24 hours, both the cultures produced higher titratable and volatile acidities and liberated more tyrosine in buffalo's milk as compared to either cow's or goat's milk. These cultures did not produce diacetyl or acetoin in different types of milk. It was further observed that the mutant was biochemically more active as compared to the parent culture. L. casei (RTS), irrespective of milk of different species. (orig.) [de
Sonwalkar, V. S.; Reddy, A.
2017-12-01
Variation in field-aligned electron and ion densities as a function of geomagnetic activity are important parameters in the physics of the thermosphere-ionosphere-magnetosphere coupling. Using whistler mode sounding from IMAGE, we report variations in field-aligned electron density and O+/H+ transition height (HT) during two periods (16-23 Aug 2005; 24 Sep-06 Oct 2005) when geomagnetic conditions were quiet (maximum Kp in the past 24 hours, Kpmax,24 ≤ 2) to moderately active (2 quiet time, during moderate geomagnetic activity: (1) O+/H+ transition height was roughly same; (2) electron density variations below HT showed no trend; (3) electron density above HT increased ( 10-40 %). The measured electron density is in agreement with in situ measurements from CHAMP (350 km) and DMSP (850 km) and past space borne (e. g., ISIS) measurements but the F2 peak density is a factor of 2 lower relative to that measured by ground ionosondes and that predicted by IRI-2012 empirical model. The measured transition height is consistent with OGO 4, Explorer 31, and C/NOFS measurements but is lower than that from IRI-2012. The observed variations in electron density at F2 peak are consistent with past work and are attributed to solar, geomagnetic, and meteorological causes [e. g. Risibeth and Mendillo, 2001; Forbes et al., 2000]. To the best of our knowledge, variations in field-aligned electron density above transition height at mid-latitudes during quiet to moderately active periods have not been reported in the past. Further investigation using physics based models (e. g., SAMI3) is required to explain the observed variations.
International Nuclear Information System (INIS)
Lui, A.T.Y.; Meng, C.
1979-01-01
The implications of southward magnetic fields at the magnetotail neutral sheet to the development of streaming anisotropy of energetic electrons and magnetospheric substorm activity are examined. Magnetic field and energetic particle measurements from the Imp 6 spacecraft, the AE index, and global auroral images from DMSP spacecraft are utilized in this study. Criteria are developed to identify events of southward magnetic fields at the neutral sheet which imply the presence of X-type magnetic neutral lines. Several features of the observations suggest that the southward magnetic fields and the implied X-type neutral lines are associated with magnetic bubbles in the neutral sheet region. It is found that the signatures of magnetic bubbles are sometimes detected in association with tailward streaming and flux enhancement of energetic electrons (47 keV< E<350keV). A cigar-shaped anisotropy in the energetic electron distribution is frequently but not always observed before the onset of tailward streaming of energetic electrons. The tailward streaming is magnetic field-aligned and occurs in the form of bursts, suggestic electrons. The tailward streaming is magnetic field-aligned and occurs in the form of bursts, suggesting that the generating process is activated somewhat quasi-periodically and is not in a steady state. Signatures of magnetic bubbles are also detected without any substantial enhancement or detectable tailward streaming of energetic electrons. By comparing Imp 6 observations with the AW index and global auroral images from DMSP spacecraft. It is found that signatures of magnetic bubbles in the neutral sheet are observed during substorms as well as during quiet geomagnetic conditions, indicating that magnetic bubbles are intrinsic features of the neutral sheet in the magnetotail regardless of substorm activity
Pavlov, Y. S.; Revina, A. A.; Souvorova, O. V.; Voropaeva, N. L.; Chekmar, D. V.; Abkhalimov, E. V.; Zavyalov, M. A.; Filippovich, V. P.
2017-12-01
Electron beam (EB) radiation technologies have been employed to increase efficiency of biologically active nanochips developed for agricultural plants seed pre-treatment with purpose of enhancing crop yield and productivity. Iron-containing nanoparticles (NPs), synthesized in reverse micelles following known radiation-chemical technique, have served as a multifunctional biologically active and phytosanitary substance of the chips. Porous chip carriers activation has been performed by EB ionization (doze 20kGy) of the active carbons (AC) prepared from agricultural waste and by-products: Jerusalem artichoke (Helianthus tuberosus) straw, rape (Brassica napus L. ssp. oleifera Metzg) straw, camelina (Camelina sativa (L.) Crantz) straw, wheat (Triticum aestivum) straw. Three methods, UV-VIS spectrophotometry, Electron Paramagnetic Resonance (EPR) spectroscopy, cyclic voltammetry (CV) have been used for process control and characterization of radiation-activated and NPs-modified ACs. The results show a notable effect of ACs activation by electron beam radiation, evidenced by FeNPs-adsorption capacity increase. Studies of the impact of Fe NPs-containing nanochip technology on enhancement of seeds germination rate and seedlings vigour suggest that reported electron beam radiation treatment techniques of the ACs from selected agricultural residues may be advantageous for industrial application.
Alves, M J; Fernandes, Â; Barreira, J C M; Lourenço, I; Fernandes, D; Moura, A; Ribeiro, A R; Salgado, J; Antonio, A; Ferreira, I C F R
2015-03-01
The effects of irradiation (gamma-rays and electron-beams), up to 10 kGy, in the antimicrobial activity of mushroom species (Boletus edulis, Hydnum repandum, Macrolepiota procera and Russula delica) differently processed (fresh, dried, freeze) were evaluated. Clinical isolates with different resistance profiles from hospitalized patients in Local Health Unit of Mirandela, Northeast of Portugal, were used as target micro-organisms. The mushrooms antimicrobial activity did not suffer significant changes that might compromise applying irradiation as a possible mushroom conservation technology. Two kGy dose (independently of using gamma-rays or electron-beams) seemed to be the most suitable choice to irradiate mushrooms. This study provides important results in antimicrobial activity of extracts prepared from irradiated mushroom species. © 2014 The Society for Applied Microbiology.
Directory of Open Access Journals (Sweden)
Assaf Shapira
Full Text Available The synthesis of inactive enzyme precursors, also known as "zymogens," serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV as a model, we designed two HCV NS3 protease-activated "zymogenized" chimeric toxins (which we denote "zymoxins". In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA and Ricin A chain (RTA, respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the "zymoxin" approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected.
Mekkes, J. R.; Le Poole, I. C.; Das, P. K.; Kammeyer, A.; Westerhof, W.
1997-01-01
Wound debridement, the removal of necrotic tissue, can be achieved with proteolytic enzymes. Recently, a new multi-enzyme preparation, krill enzyme, isolated from Antarctic shrimp-like organisms (Euphausia superba), was reported to possess powerful proteolytic activity towards protein substrates. In
Torres-Bonilla, Kristian A; Floriano, Rafael S; Schezaro-Ramos, Raphael; Rodrigues-Simioni, Léa; da Cruz-Höfling, Maria Alice
2017-06-01
Colombian colubrid snake venoms have been poorly studied. They represent a great resource of biological, ecological, toxinological and pharmacological research. We assessed some enzymatic properties and neuromuscular effects of Erythrolamprus bizona and Pseudoboa neuwiedii venoms from Colombia. Proteolytic, amidolytic and phospholipase A 2 (PLA 2 ) activities were analyzed using colorimetric assays and the neuromuscular activity was analyzed in chick biventer cervicis (BC) preparations. The venom of both species showed very low PLA 2 and amidolytic activities; however, both exhibited high proteolytic activity, which in E. bizona venom surpassed that of P. neuwiedii venom. E. bizona and P. neuwiedii venoms provoked partial neuromuscular blockade, which was more prominent in P. neuwiedii venom. E. bizona venom (30 μg/ml) induced a significant potentiation of the contracture response to exogenous ACh (110 μM), which was not accompanied by twitch height alteration, whereas the highest venom concentration (100 μg/ml) inhibited contracture responses to both ACh and KCl (40 mM). In contrast, P. neuwiedii venom (30 and 100 μg/ml) caused significant reduction in the contracture responses to exogenous ACh and KCl. The morphological analyses showed high myotoxic effects in the muscle fibers of BC incubated with either venoms; however, they are more prominent in the P. neuwiedii venom. Our results suggest that the myotoxicity of the venom of the two Colombian species can be ascribed to their high proteolytic activity. An interesting data was the potentiation of the ACh-induced contracture, but not the twitch height, caused by E. bizona venom, at a concentration that is harmless to muscle fibers integrity. This phenomenon remains to be further elucidated, and suggest that a possible involvement of post-synaptic receptors cannot be discarded. This work is a contribution to expand the knowledge on colubrid venoms; it allows envisaging that the two venoms offer
Llewellyn-Jones, C G; Hill, S L; Stockley, R A
1994-03-01
Corticosteroids are widely used in the treatment of many inflammatory conditions but the exact mode of action on neutrophil function is uncertain. Fluticasone propionate is a new topically active synthetic steroid which can be measured in body fluids and which undergoes first pass metabolism. The effects of fluticasone propionate on the function of neutrophils isolated from normal, healthy control subjects and on the chemotactic activity of sputum sol phase were assessed. Preincubation of neutrophils with fluticasone propionate reduced the chemotactic response to 10(-8) mol/l F-Met-Leu-Phe (FMLP) and to a 1:5 dilution of sputum sol phase in a dose dependent manner. Furthermore, when fluticasone propionate was added to sputum from eight patients with stable chronic obstructive bronchitis the chemotactic activity of a 1:5 dilution of the sol phase fell from a mean (SE) value of 22.2 (1.21) cells/field to 19.6 (0.89), 17.1 (0.74), and 11.9 (0.6) cells field at 1 mumol/l, 10 mumol/l, and 100 mumol/l, respectively. In further experiments fluticasone propionate preincubated with neutrophils inhibited fibronectin degradation by resting cells and by cells stimulated by FMLP (15.2% inhibition of resting cells, 5.1% inhibition of stimulated cells with 1 mumol/l fluticasone propionate, 24% and 18.7% inhibition respectively at 100 mumol/l fluticasone propionate. Fluticasone propionate had no effect on generation of superoxide anion by resting or stimulated cells. These results indicate that fluticasone propionate has a direct suppressive effect on several aspects of neutrophil function and may suggest a role for this agent in the modulation of neutrophil mediated damage to connective tissue.
SU-F-T-656: Monte Carlo Study On Air Activation Around a Medical Electron Linac
International Nuclear Information System (INIS)
Horst, F; Fehrenbacher, G; Zink, K
2016-01-01
Purpose: In high energy photon therapy, several radiation protection issues result from photonuclear reactions. The activation of air - directly by photonuclear reactions as well as indirectly by capture of photoneutrons generated inside the linac head - is a major point of concern for the medical staff. The purpose of this study was to estimate the annual effective dose to medical workers due to activated air around a medical high energy electron linac by means of Monte Carlo simulations. Methods: The treatment head of a Varian Clinac in 18 MV-X mode as well as the surrounding concrete bunker were modeled and the radiation transport was simulated using the Monte Carlo code FLUKA, starting from the primary electron striking the bremsstrahlung target. The activation yields in air from photo-disintegration of O-16 and N-14 nuclei as well as from neutron capture on Ar-40 nuclei were obtained from the simulations. The activation build-up, radioactive decay and air ventilation were studied using a mathematical model. The annual effective dose to workers was estimated by using published isotope specific conversion factors. Results: The oxygen and nitrogen activation yields were in contrast to the argon activation yield found to be field size dependent. The impact of the treatment room ventilation on the different air activation products was investigated and quantified. An estimate with very conservative assumptions gave an annual effective dose to workers of < 1 mSv/a. Conclusion: From the results of this study it can be concluded that the contribution of air activation to the radiation exposure to medical workers should be negligible in modern photon therapy, especially when it is compared to the dose due to prompt neutrons and the activation of heavy solid materials such as the jaws and the collimators inside the linac head.
SU-F-T-656: Monte Carlo Study On Air Activation Around a Medical Electron Linac
Energy Technology Data Exchange (ETDEWEB)
Horst, F [Institute of Medical Physics and Radiation Protection, University of Applied Sciences, Giessen (Germany); GSI Helmholtz Centre for Heavy Ion Research, Darmstadt (Germany); Fehrenbacher, G [GSI Helmholtz Centre for Heavy Ion Research, Darmstadt (Germany); Zink, K [Institute of Medical Physics and Radiation Protection, University of Applied Sciences, Giessen (Germany); University Hospital Giessen-Marburg, Marburg (Germany)
2016-06-15
Purpose: In high energy photon therapy, several radiation protection issues result from photonuclear reactions. The activation of air - directly by photonuclear reactions as well as indirectly by capture of photoneutrons generated inside the linac head - is a major point of concern for the medical staff. The purpose of this study was to estimate the annual effective dose to medical workers due to activated air around a medical high energy electron linac by means of Monte Carlo simulations. Methods: The treatment head of a Varian Clinac in 18 MV-X mode as well as the surrounding concrete bunker were modeled and the radiation transport was simulated using the Monte Carlo code FLUKA, starting from the primary electron striking the bremsstrahlung target. The activation yields in air from photo-disintegration of O-16 and N-14 nuclei as well as from neutron capture on Ar-40 nuclei were obtained from the simulations. The activation build-up, radioactive decay and air ventilation were studied using a mathematical model. The annual effective dose to workers was estimated by using published isotope specific conversion factors. Results: The oxygen and nitrogen activation yields were in contrast to the argon activation yield found to be field size dependent. The impact of the treatment room ventilation on the different air activation products was investigated and quantified. An estimate with very conservative assumptions gave an annual effective dose to workers of < 1 mSv/a. Conclusion: From the results of this study it can be concluded that the contribution of air activation to the radiation exposure to medical workers should be negligible in modern photon therapy, especially when it is compared to the dose due to prompt neutrons and the activation of heavy solid materials such as the jaws and the collimators inside the linac head.
Smartphone data as an electronic biomarker of illness activity in bipolar disorder
DEFF Research Database (Denmark)
Faurholt-Jepsen, Maria; Vinberg, Maj; Frost, Mads
2015-01-01
Objectives Objective methods are lacking for continuous monitoring of illness activity in bipolar disorder. Smartphones offer unique opportunities for continuous monitoring and automatic collection of real-time data. The objectives of the paper were to test the hypotheses that (i) daily electronic...... self-monitored data and (ii) automatically generated objective data collected using smartphones correlate with clinical ratings of depressive and manic symptoms in patients with bipolar disorder. Methods Software for smartphones (the MONARCA I system) that collects automatically generated objective......-monitored data (i.e., mood and activity) and scores on the YMRS. Finally, the automatically generated objective data were able to discriminate between affective states. Conclusions Automatically generated objective data and self-monitored data collected using smartphones correlate with clinically rated...
Hook, Vivian; Funkelstein, Lydiane; Wegrzyn, Jill; Bark, Steven; Kindy, Mark; Hook, Gregory
2012-01-01
Recent new findings indicate significant biological roles of cysteine cathepsin proteases in secretory vesicles for production of biologically active peptides. Notably, cathepsin L in secretory vesicles functions as a key protease for proteolytic processing of proneuropeptides (and prohormones) into active neuropeptides that are released to mediate cell-cell communication in the nervous system for neurotransmission. Moreover, cathepsin B in secretory vesicles has been recently identified as a β-secretase for production of neurotoxic β- amyloid (Aβ) peptides that accumulate in Alzheimer's disease (AD), participating as a notable factor in the severe memory loss in AD. These secretory vesicle functions of cathepsins L and B for production of biologically active peptides contrast with the well-known role of cathepsin proteases in lysosomes for the degradation of proteins to result in their inactivation. The unique secretory vesicle proteome indicates proteins of distinct functional categories that provide the intravesicular environment for support of cysteine cathepsin functions. Features of the secretory vesicle protein systems insure optimized intravesicular conditions that support the proteolytic activity of cathepsins. These new findings of recently discovered biological roles of cathepsins L and B indicate their significance in human health and disease. This article is part of a Special Issue entitled: Proteolysis 50 years after the discovery of lysosome. Copyright © 2011 Elsevier B.V. All rights reserved.
Isolation of Lactic Acid Bacteria with High Biological Activity from Local Fermented Dairy Products
Directory of Open Access Journals (Sweden)
B. Munkhtsetseg
2009-12-01
Full Text Available The thirty-two strains of lactic acid bacteria were isolated from the Mongolian traditional fermented dairy products, among them 25 strains show antimicrobial activity against test microorganisms including Escherichia coli , Staphylococcus aureus , Enterococcus faecalis , Pseudom о nas aeruginosa . Protease sensitivity assay demonstrated that the antimicrobial substances produced by isolates А 23, Т 2 are bacteriocins as their antibacterial activities were eliminated completely after treatment with protease. Identi fi cation of bacteria is being carried out. Among the isolates 22 strains show protease enzyme producing activity. The selected strains isolated from mare’s fermented milk (airag or kumis and yoghurt (tarag show the speci fi c protease activity from 7.9 μ g/ml to 11.9 μ g/ml. The strain T2, isolated from yoghurt exhibited the highest proteolytic activity.
Directory of Open Access Journals (Sweden)
Mareš Michael
2008-03-01
Full Text Available Abstract Background Ticks are vectors for a variety of viral, bacterial and parasitic diseases in human and domestic animals. To survive and reproduce ticks feed on host blood, yet our understanding of the intestinal proteolytic machinery used to derive absorbable nutrients from the blood meal is poor. Intestinal digestive processes are limiting factors for pathogen transmission since the tick gut presents the primary site of infection. Moreover, digestive enzymes may find practical application as anti-tick vaccine targets. Results Using the hard tick, Ixodes ricinus, we performed a functional activity scan of the peptidase complement in gut tissue extracts that demonstrated the presence of five types of peptidases of the cysteine and aspartic classes. We followed up with genetic screens of gut-derived cDNA to identify and clone genes encoding the cysteine peptidases cathepsins B, L and C, an asparaginyl endopeptidase (legumain, and the aspartic peptidase, cathepsin D. By RT-PCR, expression of asparaginyl endopeptidase and cathepsins B and D was restricted to gut tissue and to those developmental stages feeding on blood. Conclusion Overall, our results demonstrate the presence of a network of cysteine and aspartic peptidases that conceivably operates to digest host blood proteins in a concerted manner. Significantly, the peptidase components of this digestive network are orthologous to those described in other parasites, including nematodes and flatworms. Accordingly, the present data and those available for other tick species support the notion of an evolutionary conservation of a cysteine/aspartic peptidase system for digestion that includes ticks, but differs from that of insects relying on serine peptidases.
Lozito, Thomas P; Jackson, Wesley M; Nesti, Leon J; Tuan, Rocky S
2014-02-01
Mesenchymal stem cells (MSCs) are attractive candidates for inclusion in cell-based therapies by virtue of their abilities to home to wound sites. However, in-depth characterization of the specific effects of MSCs on their microenvironments is needed to realize their full therapeutic potentials. Furthermore, since MSCs of varying properties can be isolated from a diverse spectrum of tissues, a strategic and rational approach in MSC sourcing for a particular application has yet to be achieved. For example, MSCs that activate their proteolytic environments may promote tissue remodeling, while those from different tissue sources may inhibit proteases and promote tissue stabilization. This study attempts to address these issues by analyzing MSCs isolated from three adult tissue sources in terms of their effects on their proteolytic microenvironments. Human bone marrow, adipose, and traumatized muscle derived MSCs were compared in their soluble and cellular-associated MMP components and activity. For all types of MSCs, MMP activity associated with the cell surface, but activity levels and MMP profiles differed with tissue source. All MSC types bound exogenous active MMPs at their surfaces. MSCs were also able to activate exogenous proMMP-2 and proMMP-13. This is in marked contrast to the MSC soluble compartment, which strongly inhibited MMPs via endogenous TIMPs. The exact TIMP used to inhibit the exogenous MMP differed with MSC type. Thus, MSCs saturate their environment with both MMPs and TIMPs. Since they bind and activate MMPs at their surfaces, the net result is a very controlled pericellular localization of MMP activities by MSCs. © 2013.
Comparative analysis of the terms "electronic commerce" and "electronic business"
Kavaliauskienė, Virginija; Šarapovas, Tadas
2002-01-01
Establishing the terms that clearly and consistently describe growing and dynamic networked economy is a critical first step toward further analysis and evaluation of electronic commerce and electronic business processes. Electronic commerce is making an impact on the ways that purchasing activities are being conducted. Much of the early literature on this subject was very speculative. However, the growth of e-commerce has enabled more observations to be made of the use of electronic business...
International Nuclear Information System (INIS)
Hunt, W.A.
1983-01-01
The effect of two types of ionizing radiation was examined on active avoidance behaviour. Male Sprague-Dawley rats were trained to avoid footshock by jumping onto a retractable ledge. When irradiated with high-energy electrons or gamma photons, their performance was degraded in a dose-dependent manner. However, electrons were 1.6 times as effective as gamma photons with ED50s of 62 and 102 Gy, respectively. All animals recovered within 24 min for all doses used. The data suggest that different types of ionizing radiation may not be equivalent when assessing their effect on behaviour. (author)
The problem of large samples. An activation analysis study of electronic waste material
International Nuclear Information System (INIS)
Segebade, C.; Goerner, W.; Bode, P.
2007-01-01
Large-volume instrumental photon activation analysis (IPAA) was used for the investigation of shredded electronic waste material. Sample masses from 1 to 150 grams were analyzed to obtain an estimate of the minimum sample size to be taken to achieve a representativeness of the results which is satisfactory for a defined investigation task. Furthermore, the influence of irradiation and measurement parameters upon the quality of the analytical results were studied. Finally, the analytical data obtained from IPAA and instrumental neutron activation analysis (INAA), both carried out in a large-volume mode, were compared. Only parts of the values were found in satisfactory agreement. (author)
International Nuclear Information System (INIS)
Cheng, Jin; Ye, Feng; Dan, Guorong; Zhao, Yuanpeng; Wang, Bin; Zhao, Jiqing; Sai, Yan; Zou, Zhongmin
2016-01-01
Nitrogen mustard (NM), a bifunctional alkylating agent (BAA), contains two alkyl arms and can act as a cross-linking bridge between DNA and protein to form a DNA-protein cross-link (DPC). O 6 -methylguanine–DNA methyltransferase (MGMT), a DNA repair enzyme for alkyl adducts removal, is found to enhance cell sensitivity to BAAs and to promote damage, possibly due to its stable covalent cross-linking with DNA mediated by BAAs. To investigate MGMT-DNA cross-link (mDPC) formation and its possible dual roles in NM exposure, human bronchial epithelial cell line 16HBE was subjected to different concentrations of HN2, a kind of NM, and we found mDPC was induced by HN2 in a concentration-dependent manner, but the mRNA and total protein of MGMT were suppressed. As early as 1 h after HN2 treatment, high mDPC was achieved and the level maintained for up to 24 h. Quick total DPC (tDPC) and γ-H2AX accumulation were observed. To evaluate the effect of newly predicted protease DVC1 on DPC cleavage, we applied siRNA of MGMT and DVC1, MG132 (proteasome inhibitor), and NMS-873 (p97 inhibitor) and found that proteolysis plays a role. DVC1 was proven to be more important in the cleavage of mDPC than tDPC in a p97-dependent manner. HN2 exposure induced DVC1 upregulation, which was at least partially contributed to MGMT cleavage by proteolysis because HN2-induced mDPC level and DNA damage was closely related with DVC1 expression. Homologous recombination (HR) was also activated. Our findings demonstrated that MGMT might turn into a DNA damage promoter by forming DPC when exposed to HN2. Proteolysis, especially DVC1, plays a crucial role in mDPC repair. - Highlights: • Nitrogen mustard-induced MGMT-DNA cross-linking was detected in a living cell. • Concentration- and time-dependent manners of MGMT-DNA cross-linking were revealed. • Proteolysis played an important role in protein (MGMT)-DNA cross-linking repair. • DVC1 acts as a proteolytic enzyme in cross-linking repair in a p
Shapira, Assaf; Gal-Tanamy, Meital; Nahary, Limor; Litvak-Greenfeld, Dana; Zemel, Romy; Tur-Kaspa, Ran; Benhar, Itai
2011-01-01
The synthesis of inactive enzyme precursors, also known as “zymogens,” serves as a mechanism for regulating the execution of selected catalytic activities in a desirable time and/or site. Zymogens are usually activated by proteolytic cleavage. Many viruses encode proteases that execute key proteolytic steps of the viral life cycle. Here, we describe a proof of concept for a therapeutic approach to fighting viral infections through eradication of virally infected cells exclusively, thus limiting virus production and spread. Using the hepatitis C virus (HCV) as a model, we designed two HCV NS3 protease-activated “zymogenized” chimeric toxins (which we denote “zymoxins”). In these recombinant constructs, the bacterial and plant toxins diphtheria toxin A (DTA) and Ricin A chain (RTA), respectively, were fused to rationally designed inhibitor peptides/domains via an HCV NS3 protease-cleavable linker. The above toxins were then fused to the binding and translocation domains of Pseudomonas exotoxin A in order to enable translocation into the mammalian cells cytoplasm. We show that these toxins exhibit NS3 cleavage dependent increase in enzymatic activity upon NS3 protease cleavage in vitro. Moreover, a higher level of cytotoxicity was observed when zymoxins were applied to NS3 expressing cells or to HCV infected cells, demonstrating a potential therapeutic window. The increase in toxin activity correlated with NS3 protease activity in the treated cells, thus the therapeutic window was larger in cells expressing recombinant NS3 than in HCV infected cells. This suggests that the “zymoxin” approach may be most appropriate for application to life-threatening acute infections where much higher levels of the activating protease would be expected. PMID:21264238
Analysis of Enzymatic Activity of Matrix Metalloproteinase (MMP) by Collagen Zymography in Melanoma.
Walia, Vijay; Samuels, Yardena
2018-01-01
Protein zymography is the most commonly used technique to study the enzymatic activity of matrix metalloproteinases (MMPs) and their inhibitors. MMPs are proteolytic enzymes that promote extracellular matrix degradation. MMPs are frequently mutated in malignant melanomas as well as other cancers and are linked to increasing incidence of tumor metastasis. Substrate zymography characterizes MMP activity by their ability to degrade preferred substrates. Here we describe the collagen zymography technique to measure the active or latent form of MMPs using MMP-8 as an example, which is a frequently mutated MMP family member in malignant melanomas. The same technique can be used with the modification of substrate to detect metalloproteinase activity of other MMPs. Both wild-type and mutated forms of MMPs can be analyzed using a single gel using this method.
International Nuclear Information System (INIS)
Kholmanskij, A.S.; Zubkov, A.V.; Dyumaev, K.M.
1980-01-01
Using the theory of solvatochromy it is shown that chemically active state of spiropyran with nitro group is its highly polar electron-excitated state. On the basis of the image on the electron nature of chemically active state of spiropyran the dependence of the values of radiation yield of the coloured forms of spiropyrans upon their structure is explained
Millet, Jean Kaoru; Whittaker, Gary R.
2014-01-01
Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly identified betacoronavirus causing high morbidity and mortality in humans. The coronavirus spike (S) protein is the main determinant of viral entry, and although it was previously shown that MERS-CoV S can be activated by various proteases, the details of the mechanisms of proteolytic activation of fusion are still incompletely characterized. Here, we have uncovered distinctive characteristics of MERS-CoV S. We identify, by bioinformatics and peptide cleavage assays, two cleavage sites for furin, a ubiquitously expressed protease, which are located at the S1/S2 interface and at the S2′ position of the S protein. We show that although the S1/S2 site is proteolytically processed by furin during protein biosynthesis, the S2′ site is cleaved upon viral entry. MERS-CoV pseudovirion infection was shown to be enhanced by elevated levels of furin expression, and entry could be decreased by furin siRNA silencing. Enhanced furin activity appeared to partially override the low pH-dependent nature of MERS-CoV entry. Inhibition of furin activity was shown to decrease MERS-CoV S-mediated entry, as well as infection by the virus. Overall, we show that MERS-CoV has evolved an unusual two-step furin activation for fusion, suggestive of a role during the process of emergence into the human population. The ability of MERS-CoV to use furin in this manner, along with other proteases, may explain the polytropic nature of the virus. PMID:25288733
Electron transport in solid targets and in the active mixture of a CO2 laser amplifier
Galkowski, A.
The paper examines the use of the NIKE code for the Monte Carlo computation of the deposited energy profile and other characteristics of the absorption process of an electron beam in a solid target and the spatial distribution of primary ionization in the active mixture of a CO2 laser amplifier. The problem is considered in connection with the generation of intense electron beams and the acceleration of thin metal foils, as well as in connection with the electric discharge pumping of a CO2 laser amplifier.
Maquerlot, François; Galiacy, Stephane; Malo, Michel; Guignabert, Christophe; Lawrence, Daniel A; d'Ortho, Maria-Pia; Barlovatz-Meimon, Georgia
2006-11-01
Epithelium repair, crucial for restoration of alveolo-capillary barrier integrity, is orchestrated by various cytokines and growth factors. Among them keratinocyte growth factor plays a pivotal role in both cell proliferation and migration. The urokinase plasminogen activator (uPA) system also influences cell migration through proteolysis during epithelial repair. In addition, the complex formed by uPAR-uPA and matrix-bound plasminogen activator inhibitor type-1 (PAI-1) exerts nonproteolytic roles in various cell types. Here we present new evidence about the dual role of PAI-1 under keratinocyte growth factor stimulation using an in vitro repair model of rat alveolar epithelial cells. Besides proteolytic involvement of the uPA system, the availability of matrix-bound-PAI-1 is also required for an efficient healing. An unexpected decrease of healing was shown when PAI-1 activity was blocked. However, the proteolytic action of uPA and plasmin were still required. Moreover, immediately after wounding, PAI-1 was dramatically increased in the newly deposited matrix at the leading edge of wounds. We thus propose a dual role for PAI-1 in epithelial cell wound healing, both as a soluble inhibitor of proteolysis and also as a matrix-bound regulator of cell migration. Matrix-bound PAI-1 could thus be considered as a new member of the matricellular protein family.