The history of the tetracyclines.

Science.gov (United States)

Nelson, Mark L; Levy, Stuart B

2011-12-01

The history of the tetracyclines involves the collective contributions of thousands of dedicated researchers, scientists, clinicians, and business executives over the course of more than 60 years. Discovered as natural products from actinomycetes soil bacteria, the tetracyclines were first reported in the scientific literature in 1948. They were noted for their broad spectrum antibacterial activity and were commercialized with clinical success beginning in the late 1940s to the early 1950s. The second-generation semisynthetic analogs and more recent third-generation compounds show the continued evolution of the tetracycline scaffold toward derivatives with increased potency as well as efficacy against tetracycline-resistant bacteria, with improved pharmacokinetic and chemical properties. Their biologic activity against a wide spectrum of microbial pathogens and their uses in mammalian models of inflammation, neurodegeneration, and other biological systems indicate that the tetracyclines will continue to be successful therapeutics in infectious diseases and as potential therapeutics against inflammation-based mammalian cell diseases. © 2011 New York Academy of Sciences.

Advances in protease engineering for laundry detergents.

Science.gov (United States)

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

2015-12-25

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

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

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

2018-01-01

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

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

International Nuclear Information System (INIS)

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

2015-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2015-06-30

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

Toxicity of tetracyclines and tetracycline degradation products to environmentally relevant bacteria, including selected tetracycline-resistant bacteria

DEFF Research Database (Denmark)

Halling-Sørensen, B.; Sengeløv, G.; Tjørnelund, J.

2002-01-01

Tetracyclines used in veterinary therapy invariably will find their way as parent compound and degradation products to the agricultural field. Major degradation products formed due to the limited stability of parent tetracyclines (tetracycline, chlortetracycline, and oxytetracycline) in aqueous...... at the same concentration level as tetracycline, chlortetracycline, and oxytetracycline on both the sludge and the tetracycline-sensitive soil bacteria. Further, both 5a,6-anhydrotetracychne and 5a,6-anhydrochlortetracycline had potency on tetracycline-resistant bacteria supporting a mode of action different...

Detection of protease activity in cells and animals.

Science.gov (United States)

Verdoes, Martijn; Verhelst, Steven H L

2016-01-01

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

Role of protease-activated receptor-2 in inflammation, and its possible implications as a putative mediator of periodontitis

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M Holzhausen

2005-03-01

Full Text Available Proteinase-activated receptor-2 (PAR2 belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. This receptor is widely distributed throughout the body and seems to be importantly involved in inflammatory processes. PAR2 can be activated by serine proteases such as trypsin, mast cell tryptase, and bacterial proteases, such as gingipain produced by Porphyromonas gingivalis. This review describes the current stage of knowledge of the possible mechanisms that link PAR2 activation with periodontal disease, and proposes future therapeutic strategies to modulate the host response in the treatment of periodontitis.

Defective thrombus formation in mice lacking endogenous factor VII activating protease (FSAP).

Science.gov (United States)

Subramaniam, Saravanan; Thielmann, Ina; Morowski, Martina; Pragst, Ingo; Sandset, Per Morten; Nieswandt, Bernhard; Etscheid, Michael; Kanse, Sandip M

2015-04-01

Factor VII (FVII) activating protease (FSAP) is a circulating protease with a putative function in blood coagulation and fibrinolysis. Genetic epidemiological studies have implied a role for FSAP in carotid stenosis, stroke and thrombosis. To date, no in vivo evidence is available to support these claims. We have, for the first time, used FSAP-/- mice to define its role in thrombosis and haemostasis in vivo and to characterise the molecular mechanisms involved. FeCl3-induced arterial thrombosis in carotid and mesenteric artery revealed that the occlusion time was significantly increased in FSAP-/- mice (pendogenous FSAP impaired the formation of stable, occlusive thrombi in mice. The underlying in vivo effect of FSAP is more likely to be related to the modulation of TFPI rather than FVIIa.

Factor VII-activating protease

DEFF Research Database (Denmark)

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

2017-01-01

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

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

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Sonia eVerma

2016-04-01

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

Characterization of detergent compatible protease of a halophilic Bacillus sp. EMB9: differential role of metal ions in stability and activity.

Science.gov (United States)

Sinha, Rajeshwari; Khare, S K

2013-10-01

A moderately halophilic protease producer, Bacillus sp. strain isolated from sea water is described. The protease is purified to homogeneity by ammonium sulphate precipitation and CM cellulose chromatography. The serine protease has a molecular mass of 29 kDa. Enzymatic characterization of protease revealed K(m) 2.22 mg mL(-1), Vmax 1111.11 U mL(-1), pH optimum 9.0, t1/2 190 min at 60°C and salt optima 1% (w/v) NaCl. The protease is remarkably stable in hydrophilic and hydrophobic solvents at high concentrations. The purified preparation is unstable at room temperature. Ca(2+) ions are required for preventing this loss of activity. Interestingly, the activity and stability are modulated differentially. Whereas, divalent cation Ca(2+) are involved in maintaining stability in solution at room temperature by preventing unfolding, monovalent Na(+) and K(+) ions participate in regulating the activity and assist in refolding of the enzyme. Application of the protease is shown in efficient removal of blood stain. Copyright © 2012 Elsevier Ltd. All rights reserved.

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

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Wagner A S Judice

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

Tetracycline consumption and occurrence of tetracycline resistance in Salmonella typhimurium phage types from Danish pigs

DEFF Research Database (Denmark)

Emborg, Hanne-Dorthe; Vigre, Håkan; Jensen, Vibeke Frøkjær

2007-01-01

more than doubled at the national level from 12,000-13,000 kg of active compound in 1996-1998 to 29,000 kg of active compound in 2004. Instead, tetracycline-resistant S. Typhimurium phage types became more prevalent. This suggests that the spread of already established or new resistant clones, rather......The aims of the present study were to investigate at the farm-owner level the effect of prescribed tetracycline consumption in pigs and different Salmonella Typhimurium phage types on the probability that the S. Typhimurium was resistant to tetracycline. In this study, 1,307 isolates were included......, originating from 877 farm owners, and data were analyzed using logistic regression. The analysis showed that both the S. Typhimurium phage type (p type...

Protease activated receptors (PARS) mediation in gyroxin biological activity

International Nuclear Information System (INIS)

Silva, Jose Alberto Alves da

2009-01-01

Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH 2 , respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

Functional dissection of the alphavirus capsid protease: sequence requirements for activity.

Science.gov (United States)

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

2010-11-18

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

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

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Lai Meng-Jiun

2010-08-01

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

[Roles of protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, in modulation of exocrine gland functions].

Science.gov (United States)

Nishikawa, Hiroyuki

2006-07-01

Protease-activated receptor-2 (PAR-2), a G protein-coupled receptor, is activated by proteolytic unmasking of the N-terminal extracellular tethered ligand that presumably binds to the extracellular loop 2 of the receptor itself. PAR-2 is widely distributed in the mammalian body and plays various roles in biological events in the cardiovascular, respiratory, alimentary, and central neurons systems. PAR-2-activating peptides administered systemically to mice and rats trigger prompt salivation in vivo. In an in vitro study, PAR-2 agonists including the endogenous PAR-2 activator trypsin induce secretion of amylase and mucin from isolated rat parotid glands and sublingual glands, respectively. PAR-2-activating peptides administered systemically also modulate pancreatic exocrine secretion in vivo as well as in vitro. In the gastric mucosa, PAR-2 stimulation enhances secretion of mucus and pepsinogen and suppresses acid secretion. Tear secretion can also be caused by PAR-2-related peptides in PAR-2-dependent and -independent manners. PAR-2 thus plays a general or key role in the regulation of exocrine secretion. This review focuses on the physiologic and/or pathophysiologic roles of PAR-2 in glandular exocrine secretion. The possibility of PAR-2 as a target for drug development is also discussed.

Proteases and caspase-like activity in the yeast Saccharomyces cerevisiae.

Science.gov (United States)

Wilkinson, Derek; Ramsdale, Mark

2011-10-01

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

Non-antibacterial tetracycline formulations: clinical applications in dentistry and medicine

Directory of Open Access Journals (Sweden)

Ying Gu

2012-10-01

Full Text Available In 1983, it was first reported that tetracyclines (TCs can modulate the host response, including (but not limited to inhibition of pathologic matrix metalloproteinase (MMP activity, and by mechanisms unrelated to the antibacterial properties of these drugs. Soon thereafter, strategies were developed to generate non-antibacterial formulations (subantimicrobial-dose doxycycline; SDD and compositions (chemically modified tetracyclines; CMTs of TCs as host-modulating drugs to treat periodontal and other inflammatory diseases. This review focuses on the history and rationale for the development of: (a SDD which led to two government-approved medications, one for periodontitis and the other for acne/rosacea and (b CMTs, which led to the identification of the active site of the drugs responsible for MMP inhibition and to studies demonstrating evidence of efficacy of the most potent of these, CMT-3, as an anti-angiogenesis agent in patients with the cancer, Kaposi's sarcoma, and as a potential treatment for a fatal lung disease (acute respiratory distress syndrome; ARDS. In addition, this review discusses a number of clinical studies, some up to 2 years’ duration, demonstrating evidence of safety and efficacy of SDD formulations in humans with oral inflammatory diseases (periodontitis, pemphigoid as well as medical diseases, including rheumatoid arthritis, post-menopausal osteopenia, type II diabetes, cardiovascular diseases, and a rare and fatal lung disease, lymphangioleiomyomatosis.

Effects of eye rubbing on the levels of protease, protease activity and cytokines in tears: relevance in keratoconus.

Science.gov (United States)

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

2013-03-01

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

Role of tetracycline speciation in the bioavailability to Escherichia coli for uptake and expression of antibiotic resistance.

Science.gov (United States)

Zhang, Yingjie; Boyd, Stephen A; Teppen, Brian J; Tiedje, James M; Li, Hui

2014-05-06

Tetracycline contains ionizable functional groups that manifest several species with charges at different locales and differing net charge; the fractional distribution of each species depends on pH-pKa relationship in the aqueous phase. In nature, these species interact with naturally abundant cations (e.g., Ca(2+) and Mg(2+)) to form metal-tetracycline complexes in water. In this study, we used Escherichia coli MC4100/pTGM whole-cell bioreporter to investigate tetracycline uptake from solution under varying conditions of pH, salt composition and concentration by quantifying the corresponding expression of antibiotic resistance gene. The expression of antibiotic resistance gene in the E. coli bioreporter responded linearly to intracellular tetracycline concentration. Less tetracycline entered E. coli cells at solution pH of 8.0 than at pH 6.0 or 7.0 indicating reduced bioavailability of the antibiotic at higher pH. Both Mg(2+) and Ca(2+) in solution formed metal-tetracycline complexes which reduced uptake of tetracycline by E. coli hence diminishing the bioresponse. Among the various tetracycline species present in solution, including both metal-complexed and free (noncomplexed) species, zwitterionic tetracycline was identified as the predominant species that most readily passed through the cell membrane eliciting activation of the antibiotic resistance gene in E. coli. The results indicate that the same total concentration of tetracycline in ambient solution can evoke very different expression of antibiotic resistance gene in the exposed bacteria due to differential antibiotic uptake. Accordingly, geochemical factors such as pH and metal cations can modulate the selective pressure exerted by tetracycline for development and enrichment of antibiotic resistant bacteria. We suggest that tetracycline speciation analysis should be incorporated into the risk assessment framework for evaluating environmental exposure and the corresponding development of antibiotic

Removal of antibiotics in wastewater: Effect of hydraulic and solid retention times on the fate of tetracycline in the activated sludge process.

Science.gov (United States)

Kim, Sungpyo; Eichhorn, Peter; Jensen, James N; Weber, A Scott; Aga, Diana S

2005-08-01

A study was conducted to examine the influence of hydraulic retention time (HRT) and solid retention time (SRT) on the removal of tetracycline in the activated sludge processes. Two lab-scale sequencing batch reactors (SBRs) were operated to simulate the activated sludge process. One SBR was spiked with 250 microg/L tetracycline, while the other SBR was evaluated at tetracycline concentrations found in the influent of the wastewater treatment plant (WWTP) where the activated sludge was obtained. The concentrations of tetracyclines in the influent of the WWTP ranged from 0.1 to 0.6 microg/L. Three different operating conditions were applied during the study (phase 1-HRT: 24 h and SRT: 10 days; phase 2-HRT: 7.4 h and SRT: 10 days; and phase 3-HRT: 7.4 h and SRT: 3 days). The removal efficiency of tetracycline in phase 3 (78.4 +/- 7.1%) was significantly lower than that observed in phase 1 (86.4 +/- 8.7%) and phase 2 (85.1 +/- 5.4%) at the 95% confidence level. The reduction of SRT in phase 3 while maintaining a constant HRT decreased tetracycline removal efficiency. Sorption kinetics reached equilibrium within 24 h. Batch equilibrium experiments yielded an adsorption coefficient (Kads) of 8400 +/- 500 mL/g and a desorption coefficient (Kdes) of 22 600 +/- 2200 mL/g. No evidence of biodegradation for tetracycline was observed during the biodegradability test, and sorption was found to be the principal removal mechanism of tetracycline in activated sludge.

Protease activated receptors (PARS) mediation in gyroxin biological activity; Mediacao dos receptores ativados por proteases (PARs) em atividades biologicas da giroxina

Energy Technology Data Exchange (ETDEWEB)

Silva, Jose Alberto Alves da

2009-07-01

Gyroxin is a serine protease enzyme from the South American rattlesnake (Crotalus durissus terrificus) venom; it is only partially characterized and has multiple activities. Gyroxin induces blood coagulation, blood pressure decrease and a neurotoxic behavior named barrel rotation. The mechanisms involved in this neurotoxic activity are not known. Whereas gyroxin is a member of enzymes with high potential to become a new drug with clinical applications such as thrombin, batroxobin, ancrod, tripsyn and kalicrein, it is important to find out how gyroxin works. The analysis on agarose gel electrophoresis and circular dichroism confirmed the molecules' integrity and purity. The gyroxin intravenous administration in mice proved its neurotoxicity (barrel rotation). In vivo studies employing intravital microscopy proved that gyroxin induces vasodilation with the participation of protease activated receptors (PARs), nitric oxide and Na+K+ATPase. The leukocytes' adherence and rolling counting indicated that gyroxin has no pro inflammatory activity. Gyroxin induced platelet aggregation, which was blocked by inhibitors of PAR1 and PAR4 receptors (SCH 79797 and tcY-NH{sub 2}, respectively). Finally, it was proved that the gyroxin temporarily alter the permeability of the blood brain barrier (BBB). Our study has shown that both the protease-activated receptors and nitric oxide are mediators involved in the biological activities of gyroxin. (author)

Structural changes in halophilic and non-halophilic proteases in response to chaotropic reagents.

Science.gov (United States)

Sinha, Rajeshwari; Khare, S K

2014-08-01

Halophilic enzymes have been established for their stability and catalytic abilities under harsh operational conditions. These have been documented to withstand denaturation at high temperature, pH, organic solvents, and chaotropic agents. However, this stability is modulated by salt. The present study targets an important aspect in understanding protein-urea/GdmCl interactions using proteases from halophilic Bacillus sp. EMB9 and non-halophilic subtilisin (Carlsberg) from Bacillus licheniformis as model systems. While, halophilic protease containing 1 % (w/v) NaCl (0.17 M) retained full activity towards urea (8 M), non-halophilic protease lost about 90 % activity under similar conditions. The secondary and tertiary structure were lost in non-halophilic but preserved for halophilic protein. This effect could be due to the possible charge screening and shielding of the protein surface by Ca(2+) and Na(+) ions rendering it stable against denaturation. The dialyzed halophilic protease almost behaved like the non-halophilic counterpart. Incorporation of NaCl (up to 5 %, w/v or 0.85 M) in dialyzed EMB9 protease containing urea/GdmCl, not only helped regain of proteolytic activity but also evaded denaturing action. Deciphering the basis of this salt modulated stability amidst a denaturing milieu will provide guidelines and templates for engineering stable proteins/enzymes for biotechnological applications.

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

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Vojtěch Rada

2016-01-01

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

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

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Jeffrey W Koehler

2007-02-01

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

Silica Nanofibers with Immobilized Tetracycline for Wound Dressing

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Irena Lovětinská-Šlamborová

2016-01-01

Full Text Available Local antibiotic treatment has its justification for superficial infections. The advantage of this treatment is that the antibiotic has effects on bacterial agent directly at the application site. Skin infections which are intended for the local antibiotic treatment are superficial pyoderma, some festering wounds, burns of second and third degree, infected leg ulcers, or decubitus of second and third degree. Tetracyclines are available topical antibiotics with a broad bacterial spectrum. At present, ointments containing tetracycline are also used for the treatment, which rarely can lead to skin sensitization. In this paper, a development of novel nanofibrous material with immobilized tetracycline is presented. Two different methods of immobilized tetracycline quantification onto silica nanofibers are employed. It was proven that the prevailing part of tetracycline was bound weakly by physisorption forces, while the minor part was covalently bound by NH2 groups formed by the preceding functionalization. The silica nanofibers with immobilized tetracycline are promising material for wound dressing applications due to its antibacterial activity; it was proved by tests.

Activated factor X signaling via protease-activated receptor 2 suppresses pro-inflammatory cytokine production from LPS-stimulated myeloid cells.

LENUS (Irish Health Repository)

Gleeson, Eimear M

2013-07-19

Vitamin K-dependent proteases generated in response to vascular injury and infection enable fibrin clot formation, but also trigger distinct immuno-regulatory signaling pathways on myeloid cells. Factor Xa, a protease crucial for blood coagulation, also induces protease-activated receptor-dependent cell signaling. Factor Xa can bind both monocytes and macrophages, but whether factor Xa-dependent signaling stimulates or suppresses myeloid cell cytokine production in response to Toll-like receptor activation is not known. In this study, exposure to factor Xa significantly impaired pro-inflammatory cytokine production from lipopolysaccharide-treated peripheral blood mononuclear cells, THP-1 monocytic cells and murine macrophages. Furthermore, factor Xa inhibited nuclear factor-kappa B activation in THP-1 reporter cells, requiring phosphatidylinositide 3-kinase activity for its anti-inflammatory effect. Active-site blockade, γ-carboxyglutamic acid domain truncation and a peptide mimic of the factor Xa inter-epidermal growth factor-like region prevented factor Xa inhibition of lipopolysaccharide-induced tumour necrosis factor-α release. In addition, factor Xa anti-inflammatory activity was markedly attenuated by the presence of an antagonist of protease-activated receptor 2, but not protease-activated receptor 1. The key role of protease-activated receptor 2 in eliciting factor Xa-dependent anti-inflammatory signaling on macrophages was further underscored by the inability of factor Xa to mediate inhibition of tumour necrosis factor-α and interleukin-6 release from murine bone marrow-derived protease-activated receptor 2-deficient macrophages. We also show for the first time that, in addition to protease-activated receptor 2, factor Xa requires a receptor-associated protein-sensitive low-density lipoprotein receptor to inhibit lipopolysaccharide-induced cytokine production. Collectively, this study supports a novel function for factor Xa as an endogenous, receptor

Role of the durum wheat dehydrin in the function of proteases conferring salinity tolerance in Arabidopsis thaliana transgenic lines.

Science.gov (United States)

Saibi, Walid; Zouari, Nabil; Masmoudi, Khaled; Brini, Faiçal

2016-04-01

Dehydrins are claimed to stabilize macromolecules against freezing damage, dehydration, ionic or osmotic stresses, thermal stress and re-folding yield. However, their precise function remains unknown. In this context, we report the behavior of protease activities in dehydrin transgenic Arabidopsis lines against the wild type plant under salt stress (100mM NaCl). Indeed, proteases play key roles in plants, maintaining strict protein quality control and degrading specific sets of proteins in response to diverse environmental and developmental stimuli. We proved that durum wheat DHN-5 modulates the activity of some proteases, summarized on the promotion of the Cysteinyl protease and the decrease of the Aspartyl protease activity. This fact is also upgraded in salt stress conditions. We conclude that the dehydrin transgenic context encodes salinity tolerance in transgenic lines through the modulation of the interaction not only at transcriptional level but also at protein level and also with the impact of salt stress as an endogenous and exogenous effector on some biocatalysts like proteases. Copyright © 2016 Elsevier B.V. All rights reserved.

Understanding serine proteases implications on Leishmania spp lifecycle.

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

2018-01-01

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

In vitro activity of five tetracyclines and some other antimicrobial agents against four porcine respiratory tract pathogens.

Science.gov (United States)

Pijpers, A; Van Klingeren, B; Schoevers, E J; Verheijden, J H; Van Miert, A S

1989-09-01

The minimal inhibitory concentrations (MIC) of five tetracyclines and ten other antimicrobial agents were determined for four porcine bacterial respiratory tract pathogens by the agar dilution method. For the following oxytetracycline-susceptible strains, the MIC50 ranges of the tetracyclines were: P. multocida (n = 17) 0.25-0.5 micrograms/ml; B. bronchiseptica (n = 20) 0.25-1.0 micrograms/ml; H. pleuropneumoniae (n = 20) 0.25-0.5 micrograms/ml; S. suis Type 2 (n = 20) 0.06-0.25 micrograms/ml. For 19 oxytetracycline-resistant P. multocida strains the MIC50 of the tetracyclines varied from 64 micrograms/ml for oxytetracycline to 0.5 micrograms/ml for minocycline. Strikingly, minocycline showed no cross-resistance with oxytetracycline, tetracycline, chlortetracycline and doxycycline in P. multocida and in H. pleuropneumoniae. Moreover, in susceptible strains minocycline showed the highest in vitro activity followed by doxycycline. Low MIC50 values were observed for chloramphenicol, ampicillin, flumequine, ofloxacin and ciprofloxacin against P. multocida and H. pleuropneumoniae. B. bronchiseptica was moderately susceptible or resistant to these compounds. As expected tiamulin, lincomycin, tylosin and spiramycin were not active against H. pleuropneumoniae. Except for flumequine, the MIC50 values of nine antimicrobial agents were low for S. suis Type 2. Six strains of this species showed resistance to the macrolides and lincomycin.

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I.

Science.gov (United States)

Le, Tien; Lee, Hak Jin; Jin, Hyung Jong

2015-08-15

A method was developed to eliminate the proteases contaminating commercial DNase I, which can cause degradation of target protein during the purification process. Bio Basic DNase stock solution (in Tris-HCl buffer [pH 8.0] containing 5mM CaCl2) was first incubated at 50 °C to generate autolysis of proteases and zymogens, leading to a significant reduction in protease activity while preserving DNase activity. The residual protease activity was completely inhibited by further incubation with 2mM PMSF (phenylmethylsulfonyl fluoride) or 2× S8830 inhibitor cocktail. This approach could be readily applicable to eliminate the protease activity in any DNase products or during the preparation of commercial DNase. Copyright © 2015 Elsevier Inc. All rights reserved.

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines

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Conor M. Henry

2016-02-01

Full Text Available Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ∼500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis.

Influence of montmorillonite on antimicrobial activity of tetracycline and ciprofloxacin

Science.gov (United States)

Lv, Guocheng; Pearce, Cody W.; Gleason, Andrea; Liao, Libing; MacWilliams, Maria P.; Li, Zhaohui

2013-11-01

Antibiotics are used not only to fight infections and inhibit bacterial growth, but also as growth promotants in farm livestock. Farm runoff and other farm-linked waste have led to increased antibiotic levels present in the environment, the impact of which is not completely understood. Soil, more specifically clays, that the antibiotic contacts may alter its effectiveness against bacteria. In this study a swelling clay mineral montmorillonite was preloaded with antibiotics tetracycline and ciprofloxacin at varying concentrations and bioassays were conducted to examine whether the antibiotics still inhibited bacterial growth in the presence of montmorillonite. Escherichia coli was incubated with montmorillonite or antibiotic-adsorbed montmorillonite, and then the number of viable bacteria per mL was determined. The antimicrobial activity of tetracycline was affected in the presence of montmorillonite, as the growth of non-resistant bacteria was still found even when extremely high TC doses were used. Conversely, in the presence of montmorillonite, ciprofloxacin did inhibit E. coli bacterial growth at high concentrations. These results suggest that the effectiveness of antimicrobial agents in clayey soils depends on the amount of antibiotic substance present, and on the interactions between the antibiotic and the clays in the soil, as well.

Differential Top10 promoter regulation by six tetracycline analogues in plant cells

Science.gov (United States)

Love, John; Allen, George C.; Gatz, Christiane; Thompson, William F.; Brown, C. S. (Principal Investigator)

2002-01-01

The effects of five tetracycline analogues, anhydrotetracycline, doxycycline, minocycline, oxytetracycline, and tetracycline, on Top10 promoter activity in NT1 tobacco tissue culture cells have been analysed. The concentration that repressed Top10 promoter activity, the level of transgene repression and the kinetics of transgene de-repression were determined for each analogue, and could not be predicted from in vitro binding affinity to the tetracycline repressor or from comparison with animal cells. Doxycycline had the most potent effect on the Top10 promoter and completely inhibited transgene expression at 4 nmol l(-1). Tetracycline was the most versatile of the analogues tested; tetracycline inhibited the Top10 promoter at 10 nmol l(-1) and was easily washed out to restore Top10-driven expression in 12-24 h. A study was also made of the suitability for plant research of a novel tetracycline analogue, GR33076X. In animal cells, GR33076X de-repressed Top10 promoter activity in the presence of inhibitory concentrations of anhydrotetracycline. In NT1, it is shown that GR 33076X can antagonize repression of the Top10 promoter in the presence of tetracycline, but not of anhydrotetracycline or of doxycycline. Different tetracycline analogues can therefore be used to regulate the Top10 promoter in plant cells and this property may be exploited in planning an optimum course of transgene regulation.

[Tetracyclines, sulfonamides and metronidazole].

Science.gov (United States)

Pérez-Trallero, Emilio; Iglesias, Luis

2003-11-01

Tetracyclines form a group of natural and semisynthetic products that acts inhibiting the bacterial protein synthesis. They are bacteriostatic agents, exhibiting activity against a wide range of organisms, but they are at the present of limited use because of their acquired resistance. Doxycycline is currently the most frequently used tetracycline in human medicine and it is included in the List of Essential Medicines of the World Health Organization. Sulfonamides are synthetic, broad-spectrum bacteriostatic antibiotics. They were the first effective systemic antimicrobial agents. Their mode of action is based on the inhibition of DNA synthesis. Due to their toxicity and high adquired resistance their use is currently very low. Metronidazole is the main compound of 5-nitroimidazole family. It is a very active bactericidal antibiotic against anaerobic and some microaerophilic bacteria and it is still very useful in the treatment of bacterian and parasitic infections.

Rapid Detection of Thrombin and Other Protease Activity Directly in Whole Blood

Science.gov (United States)

Yu, Johnson Chung Sing

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

Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1

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Zhou Xiangdong

2008-05-01

Full Text Available Abstract Presenilin (PS1 or PS2 is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP. PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ42 production compared to the wild type (wt, and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ38 and Aβ42, but to reduce levels of Aβ39, showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing.

Comparative characterization of protease activity in cultured spotted rose snapper juveniles (Lutjanus guttatus

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Emyr Peña

2015-09-01

Full Text Available Partial characterizations of digestive proteases were studied in three life stages of spotted rose snapper: early (EJ, middle (MJ and late juvenile (LJ with corresponding average weights of 21.3 ± 2.6 g (3 months after hatching, MAH, 190 ± 4.4 g (7 MAH, and 400 ± 11.5 g (12 MAH. At sampling points, the digestive tract was dissected into the stomach (St, pyloric caeca (PC, and the intestine in three sections (proximal (PI, middle (MI and distal intestine (DI. The effect of pH and temperature and specific inhibitors were evaluated for acid and alkaline proteases. Total acid and alkaline protease activity showed a tendency to increase with juvenile life stage of fish while trypsin activity decreased. Differences were found in acid and alkaline protease activities at different pH and temperatures during juvenile stages. Pepstatin A inhibited total activity in the stomach extract in all juvenile stages. Activity in total alkaline protease inhibition was significantly higher in EJ using TLCK, PMSF, SBTI, Phen and Ovo than in MJ and LJ, while no significant differences were found with TPCK inhibition. Therefore increases in protease activities with fish growth through juvenile stages in which a substitution or diversification in the type of alkaline enzymes exist. These results lead a better comprehension of changes in digestive potential of Lutjanidae fish.

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...

The m-AAA Protease Associated with Neurodegeneration Limits MCU Activity in Mitochondria.

Science.gov (United States)

König, Tim; Tröder, Simon E; Bakka, Kavya; Korwitz, Anne; Richter-Dennerlein, Ricarda; Lampe, Philipp A; Patron, Maria; Mühlmeister, Mareike; Guerrero-Castillo, Sergio; Brandt, Ulrich; Decker, Thorsten; Lauria, Ines; Paggio, Angela; Rizzuto, Rosario; Rugarli, Elena I; De Stefani, Diego; Langer, Thomas

2016-10-06

Mutations in subunits of mitochondrial m-AAA proteases in the inner membrane cause neurodegeneration in spinocerebellar ataxia (SCA28) and hereditary spastic paraplegia (HSP7). m-AAA proteases preserve mitochondrial proteostasis, mitochondrial morphology, and efficient OXPHOS activity, but the cause for neuronal loss in disease is unknown. We have determined the neuronal interactome of m-AAA proteases in mice and identified a complex with C2ORF47 (termed MAIP1), which counteracts cell death by regulating the assembly of the mitochondrial Ca 2+ uniporter MCU. While MAIP1 assists biogenesis of the MCU subunit EMRE, the m-AAA protease degrades non-assembled EMRE and ensures efficient assembly of gatekeeper subunits with MCU. Loss of the m-AAA protease results in accumulation of constitutively active MCU-EMRE channels lacking gatekeeper subunits in neuronal mitochondria and facilitates mitochondrial Ca 2+ overload, mitochondrial permeability transition pore opening, and neuronal death. Together, our results explain neuronal loss in m-AAA protease deficiency by deregulated mitochondrial Ca 2+ homeostasis. Copyright © 2016 Elsevier Inc. All rights reserved.

Neutrophil-Derived Proteases Escalate Inflammation through Activation of IL-36 Family Cytokines.

Science.gov (United States)

Henry, Conor M; Sullivan, Graeme P; Clancy, Danielle M; Afonina, Inna S; Kulms, Dagmar; Martin, Seamus J

2016-02-02

Recent evidence has strongly implicated the IL-1 family cytokines IL-36α, IL-36β, and IL-36γ as key initiators of skin inflammation. Similar to the other members of the IL-1 family, IL-36 cytokines are expressed as inactive precursors and require proteolytic processing for activation; however, the responsible proteases are unknown. Here, we show that IL-36α, IL-36β, and IL-36γ are activated differentially by the neutrophil granule-derived proteases cathepsin G, elastase, and proteinase-3, increasing their biological activity ~500-fold. Active IL-36 promoted a strong pro-inflammatory signature in primary keratinocytes and was sufficient to perturb skin differentiation in a reconstituted 3D human skin model, producing features resembling psoriasis. Furthermore, skin eluates from psoriasis patients displayed significantly elevated cathepsin G-like activity that was sufficient to activate IL-36β. These data identify neutrophil granule proteases as potent IL-36-activating enzymes, adding to our understanding of how neutrophils escalate inflammatory reactions. Inhibition of neutrophil-derived proteases may therefore have therapeutic benefits in psoriasis. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Earthworm Protease

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Rong Pan

2010-01-01

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

Earthworm Protease

International Nuclear Information System (INIS)

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

2010-01-01

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

Different contributions of HtrA protease and chaperone activities to Campylobacter jejuni stress tolerance and physiology

DEFF Research Database (Denmark)

Bæk, Kristoffer Torbjørn; Vegge, Christina Skovgaard; Skórko-Glonek, Joanna

2011-01-01

activity is sufficient for growth at high temperature or oxidative stress, whereas the HtrA protease activity is only essential at conditions close to the growth limit for C. jejuni. However, the protease activity was required to prevent induction of the cytoplasmic heat-shock response even at optimal......The microaerophilic bacterium Campylobacter jejuni is the most common cause of bacterial food-borne infections in the developed world. Tolerance to environmental stress relies on proteases and chaperones in the cell envelope such as HtrA and SurA. HtrA displays both chaperone and protease activity......, but little is known about how each of these activities contributes to stress tolerance in bacteria. In vitro experiments showed temperature dependent protease and chaperone activities of C. jejuni HtrA. A C. jejuni mutant lacking only the protease activity of HtrA was used to show that the HtrA chaperone...

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

Science.gov (United States)

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

2018-02-01

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

Monitoring tetracycline through a solid-state nanopore sensor

Science.gov (United States)

Zhang, Yuechuan; Chen, Yanling; Fu, Yongqi; Ying, Cuifeng; Feng, Yanxiao; Huang, Qimeng; Wang, Chao; Pei, De-Sheng; Wang, Deqiang

2016-06-01

Antibiotics as emerging environmental contaminants, are widely used in both human and veterinary medicines. A solid-state nanopore sensing method is reported in this article to detect Tetracycline, which is based on Tet-off and Tet-on systems. rtTA (reverse tetracycline-controlled trans-activator) and TRE (Tetracycline Responsive Element) could bind each other under the action of Tetracycline to form one complex. When the complex passes through nanopores with 8 ~ 9 nanometers in diameter, we could detect the concentrations of Tet from 2 ng/mL to 2000 ng/mL. According to the Logistic model, we could define three growth zones of Tetracycline for rtTA and TRE. The slow growth zone is 0-39.5 ng/mL. The rapid growth zone is 39.5-529.7 ng/mL. The saturated zone is > 529.7 ng/mL. Compared to the previous methods, the nanopore sensor could detect and quantify these different kinds of molecule at the single-molecule level.

A Trichomonas vaginalis Rhomboid Protease and Its Substrate Modulate Parasite Attachment and Cytolysis of Host Cells

Science.gov (United States)

Riestra, Angelica M.; Gandhi, Shiv; Sweredoski, Michael J.; Moradian, Annie; Hess, Sonja; Urban, Sinisa; Johnson, Patricia J.

2015-01-01

Trichomonas vaginalis is an extracellular eukaryotic parasite that causes the most common, non-viral sexually transmitted infection worldwide. Although disease burden is high, molecular mechanisms underlying T. vaginalis pathogenesis are poorly understood. Here, we identify a family of putative T. vaginalis rhomboid proteases and demonstrate catalytic activity for two, TvROM1 and TvROM3, using a heterologous cell cleavage assay. The two T. vaginalis intramembrane serine proteases display different subcellular localization and substrate specificities. TvROM1 is a cell surface membrane protein and cleaves atypical model rhomboid protease substrates, whereas TvROM3 appears to localize to the Golgi apparatus and recognizes a typical model substrate. To identify TvROM substrates, we interrogated the T. vaginalis surface proteome using both quantitative proteomic and bioinformatic approaches. Of the nine candidates identified, TVAG_166850 and TVAG_280090 were shown to be cleaved by TvROM1. Comparison of amino acid residues surrounding the predicted cleavage sites of TvROM1 substrates revealed a preference for small amino acids in the predicted transmembrane domain. Over-expression of TvROM1 increased attachment to and cytolysis of host ectocervical cells. Similarly, mutations that block the cleavage of a TvROM1 substrate lead to its accumulation on the cell surface and increased parasite adherence to host cells. Together, these data indicate a role for TvROM1 and its substrate(s) in modulating attachment to and lysis of host cells, which are key processes in T. vaginalis pathogenesis. PMID:26684303

Tetracycline hypersensitivity of an ezrA mutant links GalE and TseB (YpmB to cell division

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Pamela eGamba

2015-04-01

Full Text Available Cell division in bacteria is initiated by the polymerization of FtsZ into a ring-like structure at midcell that functions as a scaffold for the other cell division proteins. In Bacillus subtilis, the conserved cell division protein EzrA is involved in modulation of Z-ring formation and coordination of septal peptidoglycan synthesis. Here, we show that an ezrA mutant is hypersensitive to tetracycline, even when the tetracycline efflux pump TetA is present. This effect is not related to the protein translation inhibiting activity of tetracycline. Overexpression of FtsL suppresses this phenotype, which appears to be related to the intrinsic low FtsL levels in an ezrA mutant background. A transposon screen indicated that the tetracycline effect can also be suppressed by overproduction of the cell division protein ZapA. In addition, tetracycline sensitivity could be suppressed by transposon insertions in galE and the unknown gene ypmB, which was renamed tseB (tetracycline sensitivity suppressor of ezrA. GalE is an epimerase using UDP-glucose and UDP-N-acetylglucosamine as substrate. Deletion of this protein bypasses the synthetic lethality of zapA ezrA and sepF ezrA double mutations, indicating that GalE influences cell division. The transmembrane protein TseB contains an extracytoplasmic peptidase domain, and a GFP fusion shows that the protein is enriched at cell division sites. A tseB deletion causes a shorter cell phenotype, indicating that TseB plays a role in cell division. Why a deletion of ezrA renders B. subtilis cells hypersensitive for tetracycline remains unclear. We speculate that this phenomenon is related to the tendency of tetracycline analogues to accumulate into the lipid bilayer, which may destabilize certain membrane proteins.

COL-3, a chemically modified tetracycline, inhibits lipopolysaccharide-induced microglia activation and cytokine expression in the brain.

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Rawan Abdulhameed Edan

Full Text Available Microglia activation results in release of proinflammatory molecules including cytokines, which contribute to neuronal damage in the central nervous system (CNS if not controlled. Tetracycline antibiotics such as minocycline inhibit microglial activation and cytokine expression during CNS inflammation. In the present study we found that administration of chemically modified tetracycline-3 (COL-3, inhibits lipopolysaccharide (LPS-induced microglial and p38 MAPK activation, as well as the increase in TNF-α, but not IL-1β expression, in the brains of BALB/c mice. COL-3 has been described to have no antibacterial activity. We observed that COL-3 had no activity against a Gram-negative bacteria, Escherichia coli; however surprisingly, COL-3 had antibacterial activity against a Gram-positive bacteria Staphylococcus aureus, with a minimum inhibitory concentration of 1 mg/ml. Our data show that COL-3 has some antibacterial activity against S. aureus, inhibits LPS-induced neuroinflammation, and displays potential as a therapeutic agent for treatment of conditions involving CNS inflammation.

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

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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.

Thrombin like activity of Asclepias curassavica L. latex: action of cysteine proteases.

Science.gov (United States)

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.

Transformation kinetics and pathways of tetracycline antibiotics with manganese oxide

Energy Technology Data Exchange (ETDEWEB)

Wanru, Chen [School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332 (United States); Huang, Ching-Hua [School of Civil and Environmental Engineering, Georgia Institute of Technology, Atlanta, GA 30332 (United States)

2011-05-15

Tetracycline antibiotics including tetracycline (TTC), oxytetracycline (OTC) and chlorotetracycline (CTC) undergo rapid transformation to yield various products in the presence of MnO{sub 2} at mild conditions (pH 4-9 and 22 {sup o}C). Reaction rates follow the trend of CTC > TTC > OTC, and are affected by pH and complexation of TCs with Mg{sup 2+} or Ca{sup 2+}. Experimental results of TTC indicate that MnO{sub 2} promotes isomerization at the C ring to form iso-TTC and oxidizes the phenolic-diketone and tricarbonylamide groups, leading to insertion of up to 2 O most likely at the C9 and C2 positions. In contrast, reactions of OTC with MnO{sub 2} generate little iso-OTC, but occur mainly at the A ring's dimethylamine group to yield N-demethylated products. CTC yields the most complicated products upon reactions with MnO{sub 2}, encompassing transformation patterns observed with both TTC and OTC. The identified product structures suggest lower antibacterial activity than that of the parent tetracyclines. - Highlights: > Tetracyclines transform rapidly by MnO{sub 2} to yield complicated products. > Isomerized, (hydr)oxygenated and N-demethylated products are formed. > Transformation product structures may suggest lowered antibacterial activity. - The complex transformation pathways of three popular tetracycline antibiotics (tetracycline, oxytetracycline and chlorotetracycline) with MnO{sub 2} under environmental conditions are systematically evaluated and elucidated.

Transformation kinetics and pathways of tetracycline antibiotics with manganese oxide

International Nuclear Information System (INIS)

Chen Wanru; Huang, Ching-Hua

2011-01-01

Tetracycline antibiotics including tetracycline (TTC), oxytetracycline (OTC) and chlorotetracycline (CTC) undergo rapid transformation to yield various products in the presence of MnO 2 at mild conditions (pH 4-9 and 22 o C). Reaction rates follow the trend of CTC > TTC > OTC, and are affected by pH and complexation of TCs with Mg 2+ or Ca 2+ . Experimental results of TTC indicate that MnO 2 promotes isomerization at the C ring to form iso-TTC and oxidizes the phenolic-diketone and tricarbonylamide groups, leading to insertion of up to 2 O most likely at the C9 and C2 positions. In contrast, reactions of OTC with MnO 2 generate little iso-OTC, but occur mainly at the A ring's dimethylamine group to yield N-demethylated products. CTC yields the most complicated products upon reactions with MnO 2 , encompassing transformation patterns observed with both TTC and OTC. The identified product structures suggest lower antibacterial activity than that of the parent tetracyclines. - Highlights: → Tetracyclines transform rapidly by MnO 2 to yield complicated products. → Isomerized, (hydr)oxygenated and N-demethylated products are formed. → Transformation product structures may suggest lowered antibacterial activity. - The complex transformation pathways of three popular tetracycline antibiotics (tetracycline, oxytetracycline and chlorotetracycline) with MnO 2 under environmental conditions are systematically evaluated and elucidated.

Stochastic simulations of the tetracycline operon

Science.gov (United States)

2011-01-01

Background The tetracycline operon is a self-regulated system. It is found naturally in bacteria where it confers resistance to antibiotic tetracycline. Because of the performance of the molecular elements of the tetracycline operon, these elements are widely used as parts of synthetic gene networks where the protein production can be efficiently turned on and off in response to the presence or the absence of tetracycline. In this paper, we investigate the dynamics of the tetracycline operon. To this end, we develop a mathematical model guided by experimental findings. Our model consists of biochemical reactions that capture the biomolecular interactions of this intriguing system. Having in mind that small biological systems are subjects to stochasticity, we use a stochastic algorithm to simulate the tetracycline operon behavior. A sensitivity analysis of two critical parameters embodied this system is also performed providing a useful understanding of the function of this system. Results Simulations generate a timeline of biomolecular events that confer resistance to bacteria against tetracycline. We monitor the amounts of intracellular TetR2 and TetA proteins, the two important regulatory and resistance molecules, as a function of intrecellular tetracycline. We find that lack of one of the promoters of the tetracycline operon has no influence on the total behavior of this system inferring that this promoter is not essential for Escherichia coli. Sensitivity analysis with respect to the binding strength of tetracycline to repressor and of repressor to operators suggests that these two parameters play a predominant role in the behavior of the system. The results of the simulations agree well with experimental observations such as tight repression, fast gene expression, induction with tetracycline, and small intracellular TetR2 amounts. Conclusions Computer simulations of the tetracycline operon afford augmented insight into the interplay between its molecular

Stochastic simulations of the tetracycline operon

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Kaznessis Yiannis N

2011-01-01

Full Text Available Abstract Background The tetracycline operon is a self-regulated system. It is found naturally in bacteria where it confers resistance to antibiotic tetracycline. Because of the performance of the molecular elements of the tetracycline operon, these elements are widely used as parts of synthetic gene networks where the protein production can be efficiently turned on and off in response to the presence or the absence of tetracycline. In this paper, we investigate the dynamics of the tetracycline operon. To this end, we develop a mathematical model guided by experimental findings. Our model consists of biochemical reactions that capture the biomolecular interactions of this intriguing system. Having in mind that small biological systems are subjects to stochasticity, we use a stochastic algorithm to simulate the tetracycline operon behavior. A sensitivity analysis of two critical parameters embodied this system is also performed providing a useful understanding of the function of this system. Results Simulations generate a timeline of biomolecular events that confer resistance to bacteria against tetracycline. We monitor the amounts of intracellular TetR2 and TetA proteins, the two important regulatory and resistance molecules, as a function of intrecellular tetracycline. We find that lack of one of the promoters of the tetracycline operon has no influence on the total behavior of this system inferring that this promoter is not essential for Escherichia coli. Sensitivity analysis with respect to the binding strength of tetracycline to repressor and of repressor to operators suggests that these two parameters play a predominant role in the behavior of the system. The results of the simulations agree well with experimental observations such as tight repression, fast gene expression, induction with tetracycline, and small intracellular TetR2 amounts. Conclusions Computer simulations of the tetracycline operon afford augmented insight into the

Lipase and protease extraction from activated sludge

DEFF Research Database (Denmark)

Gessesse, Amare; Dueholm, Thomas; Petersen, Steffen B.

2003-01-01

of gentle and efficient enzyme extraction methods from environmental samples is very important. In this study we present a method for the extraction of lipases and proteases from activated sludge using the non-ionic detergent Triton X-100, EDTA, and cation exchange resin (CER), alone or in combination...

Two optically active molybdenum disulfide quantum dots as tetracycline sensors

Energy Technology Data Exchange (ETDEWEB)

Wang, Zhuosen; Lin, Jintai [School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Gao, Jinwei [Institute for Advanced Materials, Academy of Advanced Optoelectronics, South China Normal University, Guangzhou 510006 (China); Wang, Qianming, E-mail: qmwang@scnu.edu.cn [Key Laboratory of Theoretical Chemistry of Environment, Ministry of Education, School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); School of Chemistry and Environment, South China Normal University, Guangzhou 510006 (China); Guangzhou Key Laboratory of Materials for Energy Conversion and Storage, 510006 (China)

2016-08-01

In this work, we use the hydrothermal method to develop two luminescent MoS{sub 2} quantum dots (QDs) from L-cysteine and glutathione as sulfur precursors. The special blue emissions give rise to an instantaneous determination of tetracycline (TC) through the quenching of its luminescence. The accessibility of the optical materials and recognition mechanism have been extensively studied. This strategy demonstrated that MoS{sub 2} could act as a new platform for anchoring bioactive species or particular functional moieties. - Highlights: • MoS{sub 2} nanostructures with water solubility have been fabricated. • Blue emission has been achieved. • It displays selective detection to tetracyclines in water.

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

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Timmerman J André B

2006-03-01

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

Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia.

Science.gov (United States)

Pawlinski, Rafal; Pedersen, Brian; Schabbauer, Gernot; Tencati, Michael; Holscher, Todd; Boisvert, William; Andrade-Gordon, Patricia; Frank, Rolf Dario; Mackman, Nigel

2004-02-15

Sepsis is associated with a systemic activation of coagulation and an excessive inflammatory response. Anticoagulants have been shown to inhibit both coagulation and inflammation in sepsis. In this study, we used both genetic and pharmacologic approaches to analyze the role of tissue factor and protease-activated receptors in coagulation and inflammation in a mouse endotoxemia model. We used mice expressing low levels of the procoagulant molecule, tissue factor (TF), to analyze the effects of TF deficiency either in all tissues or selectively in hematopoietic cells. Low TF mice had reduced coagulation, inflammation, and mortality compared with control mice. Similarly, a deficiency of TF expression by hematopoietic cells reduced lipopolysaccharide (LPS)-induced coagulation, inflammation, and mortality. Inhibition of the down-stream coagulation protease, thrombin, reduced fibrin deposition and prolonged survival without affecting inflammation. Deficiency of either protease activated receptor-1 (PAR-1) or protease activated receptor-2 (PAR-2) alone did not affect inflammation or survival. However, a combination of thrombin inhibition and PAR-2 deficiency reduced inflammation and mortality. These data demonstrate that hematopoietic cells are the major pathologic site of TF expression during endotoxemia and suggest that multiple protease-activated receptors mediate crosstalk between coagulation and inflammation.

Compound list: tetracycline [Open TG-GATEs

Lifescience Database Archive (English)

Full Text Available tetracycline TC 00048 ftp://ftp.biosciencedbc.jp/archive/open-tggates/LATEST/Human/...in_vitro/tetracycline.Human.in_vitro.Liver.zip ftp://ftp.biosciencedbc.jp/archive/open-tggates/LATEST/Rat/in..._vitro/tetracycline.Rat.in_vitro.Liver.zip ftp://ftp.biosciencedbc.jp/archive/open-tggates/LATEST/Rat/in_viv...o/Liver/Single/tetracycline.Rat.in_vivo.Liver.Single.zip ftp://ftp.biosciencedbc.

Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa.

Science.gov (United States)

Oh, Jungmin; Li, Xi-Hui; Kim, Soo-Kyong; Lee, Joon-Hee

2017-06-30

Protease IV (PIV), a key virulence factor of Pseudomonas aeruginosa is a secreted lysyl-endopeptidase whose expression is induced by quorum sensing (QS). We found that PIV expressed in QS mutant has severe reduction of activity in culture supernatant (CS), even though it is overexpressed to high level. PIV purified from the QS mutant (M-PIV) had much lower activity than the PIV purified from wild type (P-PIV). We found that the propeptide cleaved from prepro-PIV was co-purified with M-PIV, but never with P-PIV. Since the activity of M-PIV was restored by adding the CS of QS-positive and PIV-deficient strain, we hypothesized that the propeptide binds to and inhibits PIV, and is degraded to activate PIV by a QS-dependent factor. In fact, the CS of the QS-positive and PIV-deficient strain was able to degrade the propeptide. Since the responsible factor should be a QS-dependently expressed extracellular protease, we tested QS-dependent proteases of P. aeruginosa and found that LasB (elastase) can degrade the propeptide and activate M-PIV. We purified the propeptide of PIV and confirmed that the propeptide can bind to and inhibit PIV. We suggest that PIV is post-secretionally activated through the extracellular degradation of the propeptide by LasB, a QS-dependent protease.

Changes in protein metabolism after irradiation. Pt. 1. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat spleen after 600 R whole body x irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1975-12-01

The protease activity of cytoplasm and cell organelles of the rat spleen against spleen protein and hemoglobin as a substrate increases during a initial reaction phase of the organism on the first day after 600 R whole body X-irradiation. The alkaline protease in the cytoplasm and the acid protease in the cell organelles increase, whereas the protease activity against externally added hemoglobin as substrate decreases below the initial values. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the spleen is therefore not explained by an increased protease activity. Acid proteases appear in the cytoplasm which derive probably from the cell organelles. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

THE EFFECT OF FEEDING Lactobacillus ON GROWTH, SURVIVAL RATE AND PROTEASE ACTIVITY OF Litopenaeus vannamei

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Nunak Nafiqoh

2011-12-01

Full Text Available This study examined the effect of two Lactobacillus bacteria on protease activity and growth rate of Litopenaeus vannamei. An experiment was conducted to examine protease activity and growth rate. The experiment consisted of two treatment tanks, the first tank was provided with artemia immersed in 2.6 x 1016 cfu/mL of bacteria solution, the second tank served as the control tank. After 20 days, the L. vannamei in the tank that received Lactobacillus have significantly different in growth, survival rate and protease activity (P<0.05 compared to the control, but no significant difference between Lactobacillus casei and Lactobacillus plantarum treatments. Within the digestive organ, protease activity of hepatopancreas and stomach demonstrated significant higher activity (P<0.05 compared to the intestine.

Bacterial proteases and virulence

DEFF Research Database (Denmark)

Frees, Dorte; Brøndsted, Lone; Ingmer, Hanne

2013-01-01

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

Comparative analysis of procoagulant and fibrinogenolytic activity of crude protease fractions of turmeric species.

Science.gov (United States)

Shivalingu, B R; Vivek, H K; Nafeesa, Zohara; Priya, B S; Swamy, S Nanjunda

2015-08-22

Turmeric rhizome is a traditional herbal medicine, which has been widely used as a remedy to stop bleeding on fresh cuts and for wound healing by the rural and tribal population of India. To validate scientific and therapeutic application of turmeric rhizomes to stop bleeding on fresh cuts and its role in wound healing process. The water extracts of thoroughly scrubbed and washed turmeric rhizomes viz., Curcuma aromatica Salisb., Curcuma longa L., Curcuma caesia Roxb., Curcuma amada Roxb. and Curcuma zedoria (Christm.) Roscoe. were subjected to salting out and dialysis. The dialyzed crude enzyme fractions (CEFs) were assessed for proteolytic activity using casein as substrate and were also confirmed by caseinolytic zymography. Its coagulant activity and fibrinogenolytic activity were assessed using human citrated plasma and fibrinogen, respectively. The type of protease(s) in CEFs was confirmed by inhibition studies using specific protease inhibitors. The CEFs of C. aromatica, C. longa and C. caesia showed 1.89, 1.21 and 1.07 folds higher proteolytic activity, respectively, compared to papain. In contrast to these, C. amada and C. zedoria exhibited moderate proteolytic activity. CEFs showed low proteolytic activities compared to trypsin. The proteolytic activities of CEFs were confirmed by caseinolytic zymography. The CEFs of C. aromatica, C. longa and C. caesia showed complete hydrolysis of Aα, Bβ and γ subunits of human fibrinogen, while C. amada and C. zedoria showed partial hydrolysis. The CEFs viz., C. aromatica, C. longa, C. caesia, C. amada and C. zedoria exhibited strong procoagulant activity by reducing the human plasma clotting time from 172s (Control) to 66s, 84s 88s, 78s and 90s, respectively. The proteolytic activity of C. aromatica, C. longa, C. caesia and C. amada was inhibited (>82%) by PMSF, suggesting the possible presence of a serine protease(s). However, C. zedoria showed significant inhibition (60%) against IAA and moderate inhibition (30

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

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Rok Gaber

2013-11-01

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

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

Science.gov (United States)

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

2013-01-01

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

Tetracycline

Science.gov (United States)

... infections caused by bacteria including pneumonia and other respiratory tract infections; ; certain infections of skin, eye, lymphatic, ... tetracycline with food, especially dairy products such as milk, yogurt, cheese, and ice cream. Follow the directions ...

Antimicrobial activity of a 48-kDa protease (AMP48) from Artocarpus heterophyllus latex.

Science.gov (United States)

Siritapetawee, J; Thammasirirak, S; Samosornsuk, W

2012-01-01

Artocarpus heterophyllus (jackfruit) is a latex producing plant. Plant latex is produced from secretory cells and contains many intergradients. It also has been used in folk medicine. This study aimed to purify and characterize the biological activities of a protease from jackfruit latex. A protease was isolated and purified from crude latex of a jackfruit tree by acid precipitation and ion exchange chromatography. The proteolytic activities of protein were tested using gelatin- and casein-zymography. The molecular weight and isoelectric point (pl) of protein were analysed by SDS/12.5% PAGE and 2D-PAGE, respectively. Antimicrobial activity of protein was analysed by broth microdilution method. In addition, the antibacterial activity of protein against Pseudomonas aeruginosa ATCC 27853 was observed and measured using atomic force microscopy (AFM) technique. The purified protein contained protease activity by digesting gelatin- and casein-substrates. The protease was designated as antimicrobial protease-48 kDa or AMP48 due to its molecular mass on SDS-PAGE was approximately 48 kDa. The isoelectric point (pl) of AMP48 was approximately 4.2. In addition, AMP48 contained antimicrobial activities by it could inhibit the growths of Pseudomonas aeruginosa ATCC 27853 and clinical isolated Candida albicans at minimum inhibitory concentration (MIC) 2.2 mg/ml and Minimum microbicidal concentration (MMC) 8.8 mg/ml. AFM image also supported the antimicrobial activities of AMP48 by the treated bacterial morphology and size were altered from normal.

Comparison of metals and tetracycline as selective agents for development of tetracycline resistant bacterial communities in agricultural soil

DEFF Research Database (Denmark)

Song, Jianxiao; Rensing, Christopher; Holm, Peter Engelund

2017-01-01

Environmental selection of antibiotic resistance may be caused by either antibiotic residues or coselecting agents. Using a strictly controlled experimental design, we compared the ability of metals (Cu or Zn) and tetracycline to (co)select for tetracycline resistance in bacterial communities. Soil...... microcosms were established by amending agricultural soil with known levels of Cu, Zn, or tetracycline known to represent commonly used metals and antibiotics for pig farming. Soil bacterial growth dynamics and bacterial community-level tetracycline resistance were determined using the [(3)H......]leucine incorporation technique, whereas soil Cu, Zn, and tetracycline exposure were quantified by a panel of whole-cell bacterial bioreporters. Tetracycline resistance increased significantly in soils containing environmentally relevant levels of Cu (≥365 mg kg(-1)) and Zn (≥264 mg kg(-1)) but not in soil spiked...

Anti-trypanosomal activity of non-peptidic nitrile-based cysteine protease inhibitors.

Science.gov (United States)

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

2017-02-01

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

Protease-activated receptor 2 modulates proliferation and invasion of oral squamous cell carcinoma cells.

Science.gov (United States)

Al-Eryani, Kamal; Cheng, Jun; Abé, Tatsuya; Maruyama, Satoshi; Yamazaki, Manabu; Babkair, Hamzah; Essa, Ahmed; Saku, Takashi

2015-07-01

Based on our previous finding that protease-activated receptor 2 (PAR-2) regulates hemophagocytosis of oral squamous cell carcinoma (SCC) cells, which induces their heme oxygenase 1-dependent keratinization, we have formulated a hypothesis that PAR-2 functions in wider activities of SCC cells. To confirm this hypothesis, we investigated immunohistochemical profiles of PAR-2 in oral SCC tissues and its functional roles in cell proliferation and invasion in SCC cells in culture. The PAR-2 expression modes were determined in 48 surgical tissue specimens of oral SCC. Using oral SCC-derived cell systems, we determined both gene and protein expression levels of PAR-2. SCC cell proliferation and invasive properties were also examined in conditions in which PAR-2 was activated by the synthetic peptide SLIGRL. PAR-2 was immunolocalized in oral SCC and carcinoma in situ cells, especially in those on the periphery of carcinoma cell foci (100% of cases), but not in normal oral epithelia. Its expression at both gene and protein levels was confirmed in 3 oral SCC cell lines including ZK-1. Activation of PAR-2 induced ZK-1 cell proliferation in a dose-dependent manner. PAR-2-activated ZK-1 cells invaded faster than nonactivated ones. The expression of PAR-2 is specific to oral malignancies, and PAR-2 regulates the growth and invasion of oral SCC cells. Copyright © 2015 Elsevier Inc. All rights reserved.

Synthesis and extended activity of triazole-containing macrocyclic protease inhibitors

DEFF Research Database (Denmark)

Pehere, A.D.; Pietsch, M.; Gütschow, M.

2013-01-01

of their activity against a panel of proteases. Acyclic azidoalkyne-based aldehydes are also evaluated for comparison. The macrocyclic peptidomimetics showed considerable activity towards calpain II, cathepsin L and S, and the 20S proteasome chymotrypsin-like activity. Some of the first examples of highly potent...

Tetracycline removal from water by adsorption/bioadsorption on activated carbons and sludge-derived adsorbents.

Science.gov (United States)

Rivera-Utrilla, José; Gómez-Pacheco, Carla V; Sánchez-Polo, Manuel; López-Peñalver, Jesús J; Ocampo-Pérez, Raúl

2013-12-15

The objective of this study was to analyze the behavior of activated carbons with different chemical and textural natures in the adsorption of three tetracyclines (TCs) (tetracycline, oxytetracycline, and chlortetracycline). We also assessed the influence of the solution pH and ionic strength on the adsorption of these compounds and studied their removal by the combined use of microorganisms and activated carbon (bioadsorption). Sludge-derived materials were also used to remove TC from water. The capacity of these materials to adsorb TC was very high and was much greater than that of commercial activated carbon. This elevated adsorption capacity (512.1-672.0 mg/g) is explained by the high tendency of TC to form complex ions with some of the metal ions present in these materials. The medium pH and presence of electrolytes considerably affected TCs adsorption on commercial activated carbon. These results indicate that electrostatic adsorbent-adsorbate interactions play an important role in TC adsorption processes when conducted at pH values that produce TC deprotonation. The presence of bacteria during the TCs adsorption process decreases their adsorption/bioadsorption on the commercial activated carbon, weakening interactions between the adsorbate and the microfilm formed on the carbon surface. The adsorptive capacity was considerably lower in dynamic versus static regime, attributable to problems of TC diffusion into carbon pores and the shorter contact time between adsorbate and adsorbent. Copyright © 2013 Elsevier Ltd. All rights reserved.

Silver-Stained Fibrin Zymography: Separation of Proteases and Activity Detection Using a Single Substrate-Containing Gel.

Science.gov (United States)

Park, Chang-Su; Kang, Dae-Ook; Choi, Nack-Shick

2017-01-01

Silver-stained fibrin zymography for separation of protease bands and activity detection using a single substrate gel was designed. The method takes advantage of the nano-scale sensitivity of both zymography and silver staining. After sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) in a gel containing fibrin (protease substrate), the gel was incubated in enzyme reaction buffer and the zymogram gel was silver-stained. Bands with protease activity were stained with silver in clear areas where the protein substrate had been degraded. The molecular sizes of proteases were accurately determined.

Electrochemical aptasensor for detecting tetracycline in milk

International Nuclear Information System (INIS)

Le, Thi Hanh; Pham, Van Phuc; La, Thi Huyen; Le, Quang Huan; Phan, Thi Binh

2016-01-01

A rapid, simple and sensitive biosensor system for tetracycline detection is very important in food safety. In this paper we developed a label-free aptasensor for electrochemical detection of tetracycline. According to the electrochemical impendence spectroscopy (EIS) analysis, there was a linear relationship between the concentration of tetracycline and the electron transfer resistance from 10 to 3000 ng ml −1 of the tetracycline concentration. The detection limit was 10 ng ml −1 in 15 min detection duration. The prepared aptasensor showed a good reproducibility with an acceptable stability in tetracycline detection. The recoveries of tetracycline in spiked milk samples were in the range of 88.1%–94.2%. The aptasensor has sensitivity 98% and specificity of 100%. (paper)

Covalent glycoinositolphospholipid (GPI binding to hemoglobin is associated with insulin-activation of erythrocyte membrane protease

Directory of Open Access Journals (Sweden)

VESNA NIKETIC

2004-05-01

Full Text Available Recently, it was demonstrated that prolonged hyperinsulinism associated with hypoglycemia, both in vivo and in vitro, caused covalent glycoinositolphospholipid (GPI binding to the C termini of both hemoglobin b-chains, which resulted in the formation of a novel, hitherto unrecognized, minor hemoglobin fraction (GPI-Hb (Niketic et al., Biochem. Biophys. Res. Commun. 239 (1997 435. In this study it was demonstrated that exposure of erythrocyte membranes to insulin causes the activation of membrane protease as well as that the formation of GPI-Hb parallels its activity. It is suggested that the insulin-activated protease is able to catalyze, albeit slowly, the transpeptidation, i.e., the replacement of the carboxy-terminal amino acid(s residues of the Hb b-chains with GPI as an exogenous nucleophile. To our knowledge the present results show for the first time that insulin stimulates protease activity in erythrocyte membranes, as well as that insulin-activated protease may be involved in post-translational GPI binding to proteins.

Efficient removal of tetracycline with KOH-activated graphene from aqueous solution.

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Ma, Jie; Sun, Yiran; Yu, Fei

2017-11-01

Activated graphene absorbents with high specific surface area (SSA) were prepared by an easy KOH-activated method, and were applied in absorbing antibiotics, such as tetracycline (TC). After activation, many micropores were introduced to graphene oxide sheets, leading to higher SSA and many new oxygen-containing functional groups, which gave KOH-activated graphene excellent adsorption capacity (approx. 532.59 mg g -1 ) of TC. Further study on the adsorption mechanism showed that the Langmuir isotherm model and the pseudo-second-order kinetic model fitted with experiment data. To further understand the adsorption process, the effects of solid-liquid ratio, pH, ionic strength and coexisting ions were also investigated. The results revealed that, compared with pH and ionic strength, solid-liquid ratio and coexisting ions (Cu 2+ , CrO 4 2- ) had more significant influence over the adsorption performance. The findings provide guidance for application of KOH-activated graphene as a promising alternative adsorbent for antibiotics removal from aqueous solutions.

Electrophoretic deposition of CdS coatings and their photocatalytic activities in the degradation of tetracycline antibiotic

Energy Technology Data Exchange (ETDEWEB)

Vázquez, A., E-mail: alejandro.lqi@gmail.com [Universidad Autónoma de Nuevo León, Facultad de Ciencias Químicas, Av. Universidad S/N, San Nicolás de los Garza, 66455 Nuevo León (Mexico); Hernández-Uresti, D.B., E-mail: ing.dianahdz@gmail.com [Universidad Autónoma de Nuevo León, CICFIM–Facultad de Ciencias Físico Matemáticas, Av. Universidad S/N, San Nicolás de los Garza, 66455 Nuevo León (Mexico); Obregón, S. [Universidad Autónoma de Nuevo León, CICFIM–Facultad de Ciencias Físico Matemáticas, Av. Universidad S/N, San Nicolás de los Garza, 66455 Nuevo León (Mexico)

2016-11-15

Highlights: • CdS photocatalyst was prepared by electrophoretic deposition. • The CdS coating was used in the photodegradation of antibiotics. • O{sub 2}{sup −} and ·OH radicals were responsible for the degradation of tetracycline. - Abstract: The photocatalytic activities of CdS coatings formed by electrophoretic deposition (EPD) were evaluated through the photodegradation of an antibiotic, tetracycline. First, CdS nanoparticles were synthesized under microwave irradiation of aqueous solutions containing the cadmium and sulfur precursors at stoichiometric amounts and by using trisodium citrate as stabilizer. Microwave irradiation was carried out in a conventional microwave oven at 2.45 GHz and 1650 W of nominal power, for 60 s. The CdS nanoparticles were characterized by UV–vis spectrophotometry, photoluminescence and X-ray diffraction. Electrophoretic deposition parameters were 300 mV, 600 mV and 900 mV of applied voltage between aluminum plates separated by 1 cm. The fractal dimensions of the surfaces were evaluated by atomic force microscopy and correlated to the morphological and topographic characteristics of the coatings. The photocatalytic activity of the CdS coatings was investigated by means the photodegradation of the tetracycline antibiotic under simulated sunlight irradiation. According to the results, the photoactivity of the coatings directly depends on the concentration of the precursors and the applied voltage during the deposition. The material obtained at 600 mV showed the best photocatalytic behavior, probably due to its physical properties, such as optimum load and suitable aggregate size.

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

Energy Technology Data Exchange (ETDEWEB)

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

1987-08-01

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

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

International Nuclear Information System (INIS)

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

1987-01-01

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

Triclabendazole Effect on Protease Enzyme Activity in the Excretory- Secretory Products of Fasciola hepatica in Vitro.

Directory of Open Access Journals (Sweden)

Yosef Shrifi

2014-03-01

Full Text Available Fasciola hepatica is one of the most important helminthes parasites and triclabendazole (TCBZ is routinely used for treatment of infected people and animals. Secreted protease enzymes by the F. hepatica plays a critical role in the invasion, migration, nutrition and the survival of parasite and are key targets for novel drugs and vaccines. The aim of study was to determine the protease activity of excretory- secretory products (ESP of F. hepatica in the presence of TCBZ anthelmintic.F. hepatica helminthes were collected and cultured within RPMI 1640 [TCBZ treated (test and untreated (control] for 6 h at 37 °C. ESP of treated and control were collected, centrifuged and supernatants were stored at -20°C. Protein concentrations were measured according to Bradford method. Protease enzymes activities of ESP samples were estimated by using sigma's non-specific protease activity assay. ESP protein bands were detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE.Mean protein concentrations in control and treated of ESP samples were determined 196.1 ±14.52 and 376.4 ±28.20 μg/ml, respectively. Mean protease enzymes activities in control and treated were 0.37 ±0.1 and 0.089 ±0.03 U/ml, respectively. Significant difference between proteins concentrations and protease enzymes activities of two groups was observed (P<0.05. SDS-PAGE showed different patterns of protein bands between treated and control samples.The TCBZ reduced secreted protease enzymes activities and possibly effects on invasion, migration, nutrition and particularly survival of the parasite in the host tissues.

Vanillin selectively modulates the action of antibiotics against resistant bacteria.

Science.gov (United States)

Bezerra, Camila Fonseca; Camilo, Cicera Janaine; do Nascimento Silva, Maria Karollyna; de Freitas, Thiago Sampaio; Ribeiro-Filho, Jaime; Coutinho, Henrique Douglas Melo

2017-12-01

The treatment of infections caused by microorganisms that are resistant to antibiotics represent one of the main challenges of medicine today, especially due to the inefficacy of long-term drug therapy. In the search for new alternatives to treat these infections, many researchers have been looking for new substances derived from natural products to replace, or be used in combination with conventional antibiotics. Vanillin is a phenolic compound whose antimicrobial activity has been used in the elimination of pathogens present in fruits and vegetables. However, its antibacterial and modulating properties remain to be characterized. Therefore, this work aimed to evaluate the antibacterial activity and analyze the modulator activity of vanillin in association with conventional antibiotics. The antimicrobial activity of vanillin was evaluated using the microdilution method to determine the Minimum Inhibitory Concentration (MIC) Standard strains of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and multi-resistant strains of Escherichia coli 06, Staphylococcus aureus 10, Pseudomonas aeruginosa 24 were used in this study. The antibiotic modulating effect was analyzed by combining vanillin with Norfloxacin, Imipenem, Gentamicin, Erythromycin and Tetracycline against the following multiresistant bacteria strains: Escherichia coli 06, Staphylococcus aureus 10 and Pseudomonas aeruginosa 24. Data were analyzed using the ANOVA test of two tracks followed by the post hoc Bonferroni test. Vanillin presented CIMs ≥1024μg/mL against all tested strains demonstrating that it did not present significant antibacterial activity. However, modulated the activity of gentamicin and imipenem against S. aureus and E. coli, causing a synergistic effect, but did not affect the activity of norfloxacin, tetracycline and erythromycin against these same microorganisms. A synergistic effect was also obtained from the association of vanillin with norfloxacin against P

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

Science.gov (United States)

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

2000-10-31

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

Protease-Activated Receptor 4 (PAR4): A Promising Target for Antiplatelet Therapy.

Science.gov (United States)

Rwibasira Rudinga, Gamariel; Khan, Ghulam Jilany; Kong, Yi

2018-02-14

Cardiovascular diseases (CVDs) are currently among the leading causes of death worldwide. Platelet aggregation is a key cellular component of arterial thrombi and major cause of CVDs. Protease-activated receptors (PARs), including PAR1, PAR2, PAR3 and PAR4, fall within a subfamily of seven-transmembrane G-protein-coupled receptors (GPCR). Human platelets express PAR1 and PAR4, which contribute to the signaling transduction processes. In association with CVDs, PAR4 not only contributes to platelet activation but also is a modulator of cellular responses that serve as hallmarks of inflammation. Although several antiplatelet drugs are available on the market, they have many side effects that limit their use. Emerging evidence shows that PAR4 targeting is a safer strategy for preventing thrombosis and consequently may improve the overall cardiac safety profile. Our present review summarizes the PAR4 structural characteristics, activation mechanism, role in the pathophysiology of diseases and understanding the association of PAR4 targeting for improved cardiac protection. Conclusively, this review highlights the importance of PAR4 antagonists and its potential utility in different CVDs.

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

Tetracycline is back. Three-step tetracycline-biotin tumour targeting

International Nuclear Information System (INIS)

Salehi, N.; Lichtenstein, M.

1998-01-01

Full text: In the 1960s, investigators attempted to use radiolabelled tetracycline for the detection of tumours. This was limited by bone and gastrointestinal uptake. The monoclonal antibody Avidin Biotin technology has been used for 10 years to target tumours. We have improved a novel mechanism using three step targeting, to demonstrate tumour cells in (C57B1/6X balb-c) F1 mice with subcutaneously implanted E-3 thymoma. The three steps were (1) i.p. injection of Biotin Tetracycline conjugate (t:1) ratio, (2) 96 h later Avidin was injected, and (3) 24 h after (2) 99m Tc-CDTPA-Biotin was injected. Avidin has four high affinity (Km 10-15) Biotin binding sites, hence step (2) couples the Avidin to Tetracycline-Biotin in the tumour. The Avidin then provides a high affinity target for the otherwise rapidly urinary excreted 99m Tc-CDTPA-Biotin. Mice were sacrificed 16-24h after (3) by cervical dislocation. Biodistribution of radioactivity tumour to blood, liver, bone and stomach were: T:BL= 7.2, T:LI= 3.35, TBO= 9.65, T:ST= 0.93. The percentage of injected dose/g was T = 4.49%, BL = 0.62%. E-3 Thymoma is a rapid growing tumour. At day 1 (step 1) the tumour size was 0.45 cm, six days later (step 3) each dimension was doubled. Hence, percentage of injected dose per gram is artefactually reduced eight-fold. With a slowly growing tumour using the same method the results may be better. The conclusions reached are that Tetracycline-Biotin 3-stage method of tumour targeting is worthy of further development

Protease-activated receptor-1 impairs host defense in murine pneumococcal pneumonia: a controlled laboratory study

NARCIS (Netherlands)

Schouten, Marcel; van't Veer, Cornelis; Roelofs, Joris J. T. H.; Levi, Marcel; van der Poll, Tom

2012-01-01

Streptococcus pneumoniae is the most common causative pathogen in community-acquired pneumonia. Protease-activated receptor-1 (PAR-1) is expressed by multiple cell types present in the lungs and can be activated by various proteases generated during acute inflammation. The cellular effect of PAR-1

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

Science.gov (United States)

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

2016-12-01

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

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

International Nuclear Information System (INIS)

Mathur, Chhavi; Savithri, Handanahal S.

2012-01-01

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

Co-evolution of insect proteases and plant protease inhibitors.

Science.gov (United States)

Jongsma, Maarten A; Beekwilder, Jules

2011-08-01

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

Caspase-dependant activation of chymotrypsin-like proteases mediates nuclear events during Jurkat T cell apoptosis

International Nuclear Information System (INIS)

O'Connell, A.R.; Lee, B.W.; Stenson-Cox, C.

2006-01-01

Apoptosis involves a cascade of biochemical and morphological changes resulting in the systematic disintegration of the cell. Caspases are central mediators of this process. Supporting and primary roles for serine proteases as pro-apoptotic mediators have also been highlighted. Evidence for such roles comes largely from the use of pharmacological inhibitors; as a consequence information regarding their apoptotic function and biochemical properties has been limited. Here, we circumvented limitations associated with traditional serine protease inhibitors through use of a fluorescently labelled inhibitor of serine proteases (FLISP) that allowed for analysis of the specificity, regulation and positioning of apoptotic serine proteases within a classical apoptotic cascade. We demonstrate that staurosporine triggers a caspase-dependant induction of chymotrypsin-like activity in the nucleus of apoptotic Jurkat T cells. We show that serine protease activity is required for the generation of late stage nuclear events including condensation, fragmentation and DNA degradation. Furthermore, we reveal caspase-dependant activation of two chymotrypsin-like protein species that we hypothesize mediate cell death-associated nuclear events

Soybean P34 Probable Thiol Protease Probably Has Proteolytic Activity on Oleosins.

Science.gov (United States)

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.

MOFzyme: Intrinsic protease-like activity of Cu-MOF

Science.gov (United States)

Li, Bin; Chen, Daomei; Wang, Jiaqiang; Yan, Zhiying; Jiang, Liang; Deliang Duan; He, Jiao; Luo, Zhongrui; Zhang, Jinping; Yuan, Fagui

2014-10-01

The construction of efficient enzyme mimetics for the hydrolysis of peptide bonds in proteins is challenging due to the high stability of peptide bonds and the importance of proteases in biology and industry. Metal-organic frameworks (MOFs) consisting of infinite crystalline lattices with metal clusters and organic linkers may provide opportunities for protease mimic which has remained unknown. Herein, we report that Cu2(C9H3O6)4/3 MOF (which is well known as HKUST-1 and denoted as Cu-MOF here), possesses an intrinsic enzyme mimicking activity similar to that found in natural trypsin to bovine serum albumin (BSA) and casein. The Michaelis constant (Km) of Cu-MOF is about 26,000-fold smaller than that of free trypsin indicating a much higher affinity of BSA for Cu-MOF surface. Cu-MOF also exhibited significantly higher catalytic efficiency than homogeneous artificial metalloprotease Cu(II) complexes and could be reused for ten times without losing in its activity. Moreover, Cu-MOF was successfully used to simulate trypsinization in cell culture since it dissociated cells in culture even without EDTA.

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

DEFF Research Database (Denmark)

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

1987-01-01

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

Multiple roles of the coagulation protease cascade during virus infection.

Science.gov (United States)

Antoniak, Silvio; Mackman, Nigel

2014-04-24

The coagulation cascade is activated during viral infections. This response may be part of the host defense system to limit spread of the pathogen. However, excessive activation of the coagulation cascade can be deleterious. In fact, inhibition of the tissue factor/factor VIIa complex reduced mortality in a monkey model of Ebola hemorrhagic fever. Other studies showed that incorporation of tissue factor into the envelope of herpes simplex virus increases infection of endothelial cells and mice. Furthermore, binding of factor X to adenovirus serotype 5 enhances infection of hepatocytes but also increases the activation of the innate immune response to the virus. Coagulation proteases activate protease-activated receptors (PARs). Interestingly, we and others found that PAR1 and PAR2 modulate the immune response to viral infection. For instance, PAR1 positively regulates TLR3-dependent expression of the antiviral protein interferon β, whereas PAR2 negatively regulates expression during coxsackievirus group B infection. These studies indicate that the coagulation cascade plays multiple roles during viral infections.

Activation of Protease-Activated Receptor 2 Induces VEGF Independently of HIF-1

DEFF Research Database (Denmark)

Rasmussen, J.G.; Riis, Simone Elkjær; Frøbert, O.

2012-01-01

Human adipose stem cells (hASCs) can promote angiogenesis through secretion of proangiogenic factors such as vascular endothelial growth factor (VEGF). In other cell types, it has been shown that induction of VEGF is mediated by both protease activated receptor 2 (PAR2) and hypoxia inducible fact...

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

Science.gov (United States)

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

2012-01-01

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

[Protease activity of microflora in the oral cavity of patients with periodontitis].

Science.gov (United States)

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.

Natural inhibitors of tumor-associated proteases

International Nuclear Information System (INIS)

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

2002-01-01

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

Hormone therapy affects plasma measures of factor VII-activating protease in younger postmenopausal women

DEFF Research Database (Denmark)

Mathiasen, Jørn Sidelmann; Skouby, S.O.; Vitzthum, F.

2010-01-01

Objectives Current reviews indicate that hormone therapy (HT) has a protective role in coronary heart disease (CHD) in younger postmenopausal women, whereas HT contributes to CHD in older women Factor VII-activating protease (FSAP) is a serine protease that accumulates in unstable atherosclerotic...

Selection of suitable detergents for obtaining an active dengue protease in its natural form from E. coli.

Science.gov (United States)

Liew, Lynette Sin Yee; Lee, Michelle Yueqi; Wong, Ying Lei; Cheng, Jinting; Li, Qingxin; Kang, CongBao

2016-05-01

Dengue protease is a two-component enzyme and is an important drug target against dengue virus. The protease activity and protein stability of dengue nonstructural protein 3 (NS3) require a co-factor region from a four-span membrane protein NS2B. A natural form of dengue protease containing full-length NS2B and NS3 protease domain NS2BFL-NS3pro will be useful for dengue drug discovery. In current study, detergents that can be used for protease purification were tested. Using a water soluble protease construct, 39 detergents were selected for both NS2B and NS2BFL-NS3pro purification. The results showed that 18 detergents were able to sustain the activity of the natural dengue protease and 11 detergents could be used for NS2B purification. The results obtained in this study will be useful for biochemical and biophysical studies on dengue protease. Copyright © 2016 Elsevier Inc. All rights reserved.

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...

The pro-coagulant fibrinogenolytic serine protease isoenzymes purified from Daboia russelii russelii venom coagulate the blood through factor V activation: role of glycosylation on enzymatic activity.

Directory of Open Access Journals (Sweden)

Ashis K Mukherjee

Full Text Available Proteases from Russell's viper venom (RVV induce a variety of toxic effects in victim. Therefore, four new RVV protease isoenzymes of molecular mass 32901.044 Da, 333631.179 Da, 333571.472 Da, and 34594.776 Da, were characterized in this study. The first 10 N-terminal residues of these serine protease isoenzymes showed significant sequence homology with N-terminal sequences of snake venom thrombin-like and factor V-activating serine proteases, which was reconfirmed by peptide mass fingerprinting analysis. These proteases were found to be different from previously reported factor V activators isolated from snake venoms. These proteases showed significantly different fibrinogenolytic, BAEE-esterase and plasma clotting activities but no fibrinolytic, TAME-esterase or amidolytic activity against the chromogenic substrate for trypsin, thrombin, plasmin and factor Xa. Their Km and Vmax values towards fibrinogen were determined in the range of 6.6 to 10.5 µM and 111.0 to 125.5 units/mg protein, respectively. On the basis of fibrinogen degradation pattern, they may be classified as A/B serine proteases isolated from snake venom. These proteases contain ∼ 42% to 44% of N-linked carbohydrates by mass whereas partially deglycosylated enzymes showed significantly less catalytic activity as compared to native enzymes. In vitro these protease isoenzymes induce blood coagulation through factor V activation, whereas in vivo they provoke dose-dependent defibrinogenation and anticoagulant activity in the mouse model. At a dose of 5 mg/kg, none of these protease isoenzymes were found to be lethal in mice or house geckos, suggesting therapeutic application of these anticoagulant peptides for the prevention of thrombosis.

Protease-Mediated Suppression of DRG Neuron Excitability by Commensal Bacteria.

Science.gov (United States)

Sessenwein, Jessica L; Baker, Corey C; Pradhananga, Sabindra; Maitland, Megan E; Petrof, Elaine O; Allen-Vercoe, Emma; Noordhof, Curtis; Reed, David E; Vanner, Stephen J; Lomax, Alan E

2017-11-29

Peripheral pain signaling reflects a balance of pronociceptive and antinociceptive influences; the contribution by the gastrointestinal microbiota to this balance has received little attention. Disorders, such as inflammatory bowel disease and irritable bowel syndrome, are associated with exaggerated visceral nociceptive actions that may involve altered microbial signaling, particularly given the evidence for bacterial dysbiosis. Thus, we tested whether a community of commensal gastrointestinal bacteria derived from a healthy human donor (microbial ecosystem therapeutics; MET-1) can affect the excitability of male mouse DRG neurons. MET-1 reduced the excitability of DRG neurons by significantly increasing rheobase, decreasing responses to capsaicin (2 μm) and reducing action potential discharge from colonic afferent nerves. The increase in rheobase was accompanied by an increase in the amplitude of voltage-gated K + currents. A mixture of bacterial protease inhibitors abrogated the effect of MET-1 effects on DRG neuron rheobase. A serine protease inhibitor but not inhibitors of cysteine proteases, acid proteases, metalloproteases, or aminopeptidases abolished the effects of MET-1. The serine protease cathepsin G recapitulated the effects of MET-1 on DRG neurons. Inhibition of protease-activated receptor-4 (PAR-4), but not PAR-2, blocked the effects of MET-1. Furthermore, Faecalibacterium prausnitzii recapitulated the effects of MET-1 on excitability of DRG neurons. We conclude that serine proteases derived from commensal bacteria can directly impact the excitability of DRG neurons, through PAR-4 activation. The ability of microbiota-neuronal interactions to modulate afferent signaling suggests that therapies that induce or correct microbial dysbiosis may impact visceral pain. SIGNIFICANCE STATEMENT Commercially available probiotics have the potential to modify visceral pain. Here we show that secretory products from gastrointestinal microbiota derived from a human

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

Science.gov (United States)

Kernaghan, Gavin; Mayerhofer, Michael

2017-01-01

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

Association Between Tetracycline Consumption and Tetracycline Resistance in Escherichia coli from Healthy Danish Slaughter Pigs

DEFF Research Database (Denmark)

Vieira, Antonio; Houe, Hans; Wegener, Henrik Caspar

2009-01-01

It has been recognized that exposure to antimicrobial agents can exert a selective pressure for the emergence of antimicrobial resistance. The objective of this study was to investigate an association between the probability of isolating a tetracycline-resistant Escherichia coli isolate from the ...... this study, we can infer that tetracycline usage, the time span between last treatment and sampling date, together with herd size and the proportion of animals being treated in a herd, increase the probability of obtaining a resistant isolate.......It has been recognized that exposure to antimicrobial agents can exert a selective pressure for the emergence of antimicrobial resistance. The objective of this study was to investigate an association between the probability of isolating a tetracycline-resistant Escherichia coli isolate from...... the intestinal tract of healthy pigs and patterns of tetracycline Consumption in the herds of origin, together with other risk factors. Data oil antimicrobial resistance, antimicrobial consumption, and pig herd demographics were obtained from different Danish surveillance programs. Descriptive statistics were...

Amylase and protease inhibition activity against Fusarium verticillioides Amylase and protease inhibition activity against Fusarium verticillioides/ Atividade inibidora de amilase e protease de milho contra Fusarium verticillioides durante a germinação

Directory of Open Access Journals (Sweden)

Elisa Yoko Hirooka

2006-06-01

Full Text Available The primary maize pathogen, Fusarium verticillioides (F. moniliforme Sheldon is responsible for fumonisin production, which is harmful to human and animal health. In addition, maize can be more susceptible to fungal infection after insect attack. The activity of amylase and protease inhibitors in AG 5011 and CD 307 hybrids were determined during germination with controlled and not controlled conditions of temperature and relative humidity and, they were correlated to maize resistance against Sithophilus zeamais. The inhibitory activity during corn germination was evaluated at 0, 24, 48, 72, 96 and 168 h. Amylase and protease inhibitory activity increased during germination in both hybrids, which ranged respectively from 2.8 to 39.5 UIA/g, and 550.0 to 3633.9 UIP/g. The highest levels of inhibitory activity occurred in hybrid CD 307 in germination chamber. The biologic cycle and susceptible rate were evaluated for corn resistance test. The AG 5011 hybrid was less susceptible to S. zeamais and showed higher inhibitory activity (time 0 h, demonstrating possible relationship between resistance against the insect and inhibitory enzymes. These results indicated that maize natural defense mechanism plays an important role on phytopathogen control.Fusarium verticillioides, patógeno primário do milho, destaca-se pela produção da fumonisina, prejudicial à saúde humana e animal. Considerando que os mecanismos naturais de defesa são ferramentas promissoras no controle de fitopatógenos, avaliou-se: a atividade dos inibidores de amilase e protease presente nos híbridos de milho AG 5011 e CD 307 durante a germinação em câmara de germinação (25ºC e 90-95%UR e em casa de vegetação (sem controle de temperatura e umidade contra amilase e protease de F. verticillioides. Paralelamente, avaliou-se a resistência do milho a Sitophilus zeamais. A atividade inibitória de enzimas avaliada nos tempos 0, 24, 48, 72, 96 e 168 h, aumentou durante a germina

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

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Zahra Ghobadi Nejad

2014-01-01

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

Great improvement on tetracycline removal using ZnO rod-activated carbon fiber composite prepared with a facile microwave method

Energy Technology Data Exchange (ETDEWEB)

Tran Thi, Viet Ha; Lee, Byeong-Kyu, E-mail: bklee@ulsan.ac.kr

2017-02-15

Highlights: • ZnO rod-ACF was prepared by a method involving a microwave within only 3 min. • ZnO rods (average diameter of 0.3–0.5 μm, length of 1.0–1.5 μm) were grown on ACF. • 99% of tetracycline was degraded and 90.7% TOC was removed within 1 h under UV light. • ZnO rod-ACF achieved high performances even after three cycles of uses. - Abstract: New composite materials of activated carbon fiber (ACF) coated with zinc oxide (ZnO) were obtained by applying a green, cost-effective and rapid synthetic route using a commercial microwave oven. ZnO rods with a uniform and stable structure and an average diameter of 0.3–0.5 μm and length of 1.0–1.5 μm were achieved after only 3-min microwave treatment. The properties of ZnO were efficiently transferred to ACF, such that the resulting material, termed ZnO rod-ACF, demonstrated a promising potential as an efficient photocatalyst and simultaneously as an adsorbent. Pharmaceutical tetracycline at a concentration of 40 mg/L was used to evaluate the organic pollutant removal capacity of the synthesized materials. At pH 8, ZnO rod-ACF exhibited excellent removal capacity (over 99%) and mineralization (90.7%) of tetracycline in aqueous solution within 1 h under UV irradiation. The stability of ZnO rod-ACF was maintained and the mineralization of tetracycline was also maintained at 81.35% after multiple usage cycles. The photodegradation pathways of tetracycline were proposed based on the identified reaction intermediates.

Protease-Activated Receptor 4 (PAR4: A Promising Target for Antiplatelet Therapy

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Gamariel Rwibasira Rudinga

2018-02-01

Full Text Available Cardiovascular diseases (CVDs are currently among the leading causes of death worldwide. Platelet aggregation is a key cellular component of arterial thrombi and major cause of CVDs. Protease-activated receptors (PARs, including PAR1, PAR2, PAR3 and PAR4, fall within a subfamily of seven-transmembrane G-protein-coupled receptors (GPCR. Human platelets express PAR1 and PAR4, which contribute to the signaling transduction processes. In association with CVDs, PAR4 not only contributes to platelet activation but also is a modulator of cellular responses that serve as hallmarks of inflammation. Although several antiplatelet drugs are available on the market, they have many side effects that limit their use. Emerging evidence shows that PAR4 targeting is a safer strategy for preventing thrombosis and consequently may improve the overall cardiac safety profile. Our present review summarizes the PAR4 structural characteristics, activation mechanism, role in the pathophysiology of diseases and understanding the association of PAR4 targeting for improved cardiac protection. Conclusively, this review highlights the importance of PAR4 antagonists and its potential utility in different CVDs.

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

Directory of Open Access Journals (Sweden)

Yu-Yin Li

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

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

Science.gov (United States)

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

2012-07-01

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

Activation of the Arabidopsis membrane-bound transcription factor bZIP28 is mediated by site-2 protease, but not site-1 protease.

Science.gov (United States)

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.

Activity-Based Protein Profiling of Rhomboid Proteases in Liposomes

Czech Academy of Sciences Publication Activity Database

Wolf, E. V.; Seybold, M.; Hadravová, Romana; Stříšovský, Kvido; Verhelst, S. H. L.

2015-01-01

Roč. 16, č. 11 (2015), s. 1616-1621 ISSN 1439-4227 R&D Projects: GA MŠk(CZ) LK11206; GA MŠk LO1302 Institutional support: RVO:61388963 Keywords : activity-based protein profiling * chemical probes * inhibitors * intramembrane proteases * liposomes Subject RIV: CE - Biochemistry Impact factor: 2.850, year: 2015

Development of a radioimmunoassay of tetracycline and its derivatives

International Nuclear Information System (INIS)

Zitzewitz, A. von.

1981-01-01

A radioimmunoassay for tetracycline was developed in the context of the present work. The determination of tetracycline content in biological samples is to be made valid using tetracycline RIA. As well as the carbodiimide method, a by radioimmunologists extremely seldomly used way involving a condensation reaction between protein and haptene via the Mannich reaction was successfully applied. Antibodies were produced using a conventional immunisation method after Abraham, the other applied alternative method after Vaitukaitis et al. The general working methods had to be adapted to the tetracycline RIA. All variable parameters of the antigen-antibody bond were tested to optimize the incubation conditions of the system as well as to control the tracer for a degradation. The detection limit of RIA is 10 - 12 , the measuring range from 10 - 12 to 10 - 10 mol for tetracycline hydrochloride and rolitetracycline respectively and up to 10 - 9 mol for its derivates. The investigations for cross reactions showed a high specificity for tetracycline (100%) and its intravenously appliable pyrrolidino-methyl derivative rolitetracycline (88%). Standard curves could be drawn up using either of the two compounds tetracycline and rolitetracycline as standard. The pharmaco-kinetic behaviour of the parenteral administrable tetracycline was analyzed as example for the possible applications of the tetracycline radioimmunoassay. Parallel animal tests administring 3 H tetracycline hydrochloride were performed for radioimmunoassay reference. The possible application of tetracycline radioimmunoassay in food analysis is discussed. (orig./MG) [de

Protease-activated receptor-2 activation exaggerates TRPV1-mediated cough in guinea pigs.

Science.gov (United States)

Gatti, Raffaele; Andre, Eunice; Amadesi, Silvia; Dinh, Thai Q; Fischer, Axel; Bunnett, Nigel W; Harrison, Selena; Geppetti, Pierangelo; Trevisani, Marcello

2006-08-01

A lowered threshold to the cough response frequently accompanies chronic airway inflammatory conditions. However, the mechanism(s) that from chronic inflammation results in a lowered cough threshold is poorly understood. Irritant agents, including capsaicin, resiniferatoxin, and citric acid, elicit cough in humans and in experimental animals through the activation of the transient receptor potential vanilloid 1 (TRPV1). Protease-activated receptor-2 (PAR2) activation plays a role in inflammation and sensitizes TRPV1 in cultured sensory neurons by a PKC-dependent pathway. Here, we have investigated whether PAR2 activation exaggerates TRPV1-dependent cough in guinea pigs and whether protein kinases are involved in the PAR2-induced cough modulation. Aerosolized PAR2 agonists (PAR2-activating peptide and trypsin) did not produce any cough per se. However, they potentiated citric acid- and resiniferatoxin-induced cough, an effect that was completely prevented by the TRPV1 receptor antagonist capsazepine. In contrast, cough induced by hypertonic saline, a stimulus that provokes cough in a TRPV1-independent manner, was not modified by aerosolized PAR2 agonists. The PKC inhibitor GF-109203X, the PKA inhibitor H-89, and the cyclooxygenase inhibitor indomethacin did not affect cough induced by TRPV1 agonists, but abated the exaggeration of this response produced by PAR2 agonists. In conclusion, PAR2 stimulation exaggerates TRPV1-dependent cough by activation of diverse mechanism(s), including PKC, PKA, and prostanoid release. PAR2 activation, by sensitizing TRPV1 in primary sensory neurons, may play a role in the exaggerated cough observed in certain airways inflammatory diseases such as asthma and chronic obstructive pulmonary disease.

Dengue Virus NS2B/NS3 Protease Inhibitors Exploiting the Prime Side.

Science.gov (United States)

Lin, Kuan-Hung; Ali, Akbar; Rusere, Linah; Soumana, Djade I; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-15

The mosquito-transmitted dengue virus (DENV) infects millions of people in tropical and subtropical regions. Maturation of DENV particles requires proper cleavage of the viral polyprotein, including processing of 8 of the 13 substrate cleavage sites by dengue virus NS2B/NS3 protease. With no available direct-acting antiviral targeting DENV, NS2/NS3 protease is a promising target for inhibitor design. Current design efforts focus on the nonprime side of the DENV protease active site, resulting in highly hydrophilic and nonspecific scaffolds. However, the prime side also significantly modulates DENV protease binding affinity, as revealed by engineering the binding loop of aprotinin, a small protein with high affinity for DENV protease. In this study, we designed a series of cyclic peptides interacting with both sides of the active site as inhibitors of dengue virus protease. The design was based on two aprotinin loops and aimed to leverage both key specific interactions of substrate sequences and the entropic advantage driving aprotinin's high affinity. By optimizing the cyclization linker, length, and amino acid sequence, the tightest cyclic peptide achieved a K i value of 2.9 μM against DENV3 wild-type (WT) protease. These inhibitors provide proof of concept that both sides of DENV protease active site can be exploited to potentially achieve specificity and lower hydrophilicity in the design of inhibitors targeting DENV. IMPORTANCE Viruses of the flaviviral family, including DENV and Zika virus transmitted by Aedes aegypti , continue to be a threat to global health by causing major outbreaks in tropical and subtropical regions, with no available direct-acting antivirals for treatment. A better understanding of the molecular requirements for the design of potent and specific inhibitors against flaviviral proteins will contribute to the development of targeted therapies for infections by these viruses. The cyclic peptides reported here as DENV protease inhibitors

Antimicrobial activity of an aspartic protease from Salpichroa origanifolia fruits.

Science.gov (United States)

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

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

Science.gov (United States)

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

2013-12-01

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

Molecular characterization of protease activity in Serratia sp. strain SCBI and its importance in cytotoxicity and virulence.

Science.gov (United States)

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.

Antibacterial Activity of Extracellular Protease Isolated From an Algicolous Fungus Xylaria psidii KT30 Against Gram-Positive Bacteria

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Taufik Indarmawan

2016-04-01

Full Text Available Infectious diseases became more serious problem for public health in recent years. Although existing antibacterial drugs have been relatively effective, they do not rule out the emergence of resistance to the drug. Therefore, the intensive exploration of new bioactive compounds from natural, especially peptide compounds began in recent decades in order-handling infection. This study aimed to isolate, purify and test the potential application of Xylaria psidii KT30 extracellular protease as antibacterial agent against Gram-positive bacteria. X. psidii KT30, a marine fungus isolated from red seaweed Kappaphycus alvarezii showed antibacterial activity against Bacillus subtilis and Staphylococcus aureus. Antibacterial compounds of this fungus were predicted as a group of proteases. Extracellular protease exhibited an optimum activity when potato dextrose broth was used as cultivation medium. Furthermore, the highest activity of these proteases was found on fungal extract after day 15 of cultivation with value of 2.33 ± 0.19 U/mL. The partial purification of proteases using G-75 column chromatography resulted in 2 groups of fractions and showed protease activity based on zymogram assay. The extracellular proteases obtained from those fractions have 3 patterns of molecular mass based on sodium dodecyl sulfate–polyacrylamide gel electrophoresis which are 56.62, 89.12, 162.18 kDa.

Tetracycline residues and tetracycline resistance genes in groundwater impacted by swine production facilities

Science.gov (United States)

Mackie, R.I.; Koike, S.; Krapac, I.; Chee-Sanford, J.; Maxwell, Susan; Aminov, R.I.

2006-01-01

Antibiotics are used at therapeutic levels to treat disease; at slightly lower levels as prophylactics; and at low, subtherapeutic levels for growth promotion and improvement of feed efficiency. Over 88% of swine producers in the United States gave antimicrobials to grower/finisher pigs in feed as a growth promoter in 2000. It is estimated that ca. 75% of antibiotics are not absorbed by animals and are excreted in urine and feces. The extensive use of antibiotics in swine production has resulted in antibiotic resistance in many intestinal bacteria, which are also excreted in swine feces, resulting in dissemination of resistance genes into the environment.To assess the impact of manure management on groundwater quality, groundwater samples have been collected near two swine confinement facilities that use lagoons for manure storage and treatment. Several key contaminant indicators-including inorganic ions, antibiotics, and antibiotic resistance genes-were analyzed in groundwater collected from the monitoring wells. Chloride, ammonium, potassium, and sodium were predominant inorganic constituents in the manure samples and served as indicators of groundwater contamination. Based on these analyses, shallow groundwater has been impacted by lagoon seepage at both sites. Liquid chromatography-mass spectroscopy (LC-MS) was used to measure the dissolved concentrations of tetracycline, chlortetracycline, and oxytetracycline in groundwater and manure. Although tetracyclines were regularly used at both facilities, they were infrequently detected in manure samples and then at relatively trace concentrations. Concentrations of all tetracyclines and their breakdown products in the groundwater sampled were generally less than 0.5 ??g/L.Bacterial tetracycline resistance genes served as distinct genotypic markers to indicate the dissemination and mobility of antibiotic resistance genes that originated from the lagoons. Applying PCR to genomic DNA extracted from the lagoon and

Trueperella pyogenes isolated from dairy cows with endometritis in Inner Mongolia, China: Tetracycline susceptibility and tetracycline-resistance gene distribution.

Science.gov (United States)

Zhang, Dexian; Zhao, Jingcui; Wang, Qiuxia; Liu, Yaochuan; Tian, Chunlian; Zhao, Yujun; Yu, Lihui; Liu, Mingchun

2017-04-01

Trueperella pyogenes plays a crucial role in endometritis pathogenesis and is also associated with many infections, including metritis, mastitis, arthritis and liver abscessation, in many domestic animals. In this study, we investigated the prevalence of tetracycline resistance in T. pyogenes isolated from dairy cows with endometritis in Inner Mongolia, China, and we assessed tetracycline-resistance gene distribution among the isolates. Our results indicated that 68.7% and 62.5% of the isolates were resistant to tetracycline and doxycycline, respectively, and the rate of resistance to metacycline was 18.8%. The tetracycline resistance gene tetK was present in all isolates (n = 32), whereas the tetM gene was identified in 12.5% and 9.4% of the isolates, in the chromosome and plasmid, respectively. Strains carrying tetW were also common in the chromosome and plasmid, with abundances of 53.1% and 46.9%, respectively. However, tetO and otrA were absent in all isolates. The resistance phenotype analysis indicated that 6.3% of strains were susceptible to all tetracyclines, while 3.1% showed resistance to all tetracyclines. Copyright © 2017 Elsevier Ltd. All rights reserved.

Changes in protein metabolism after irradiation. Pt. 2. Protease activity, protease pattern, protein and free amino acids in cytoplasm and cell organelles of the rat liver after 600 R whole body X-irradiation

Energy Technology Data Exchange (ETDEWEB)

Valet, G [Max-Planck-Institut fuer Biochemie, Muenchen (F.R. Germany). Abt. fuer Experimentelle Medizin

1976-01-01

The protease activity of cytoplasm and cell organelles of the rat liver against liver protein and hemoglobin as a substrate increases during an initial reaction phase on the first day after 600 R whole body x irradiation. This is probably a consequence of the degradation of cellular debris. The protein, the protease activity and the free amino acids of the cytoplasm and the cell organelles decrease during the disease phase on day 3 and 4 after irradiation. The protein loss of the liver is therefore not explained by an increased protease activity. The protease activity and the free amino acids are increased in the cytoplasm and the cell organelles during the regeneration phase of the organism between day 15 and 18 after irradiation.

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

Energy Technology Data Exchange (ETDEWEB)

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

2002-08-01

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

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Energy Technology Data Exchange (ETDEWEB)

Pellegrini, M.C.; Zalazar, L.; Fuselli, S.L.; Ponce, A.G.

2017-07-01

American foulbrood (AFB) is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS) mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO). The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control) was observed when a low density culture was incubated with late exponential spent medium (SM) suggesting the presence of factor(s) inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%). We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis) on exoproteases, an interesting therapeutic strategy to control AFB.

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

International Nuclear Information System (INIS)

Pellegrini, M.C.; Zalazar, L.; Fuselli, S.L.; Ponce, A.G.

2017-01-01

American foulbrood (AFB) is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS) mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO). The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control) was observed when a low density culture was incubated with late exponential spent medium (SM) suggesting the presence of factor(s) inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%). We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis) on exoproteases, an interesting therapeutic strategy to control AFB.

Inhibitory action of essential oils against proteases activity of Paenibacillus larvae, the etiological agent of American Foulbrood disease

Directory of Open Access Journals (Sweden)

María C. Pellegrini

2018-02-01

Full Text Available American foulbrood (AFB is a disease affecting the larva of Apis mellifera. The etiological agent is Paenibacillus larvae, which releases metalloproteases involved in the degradation of larval tissues. Through quorum sensing (QS mechanism, bacteria are able to activate specific genes such as virulence factors. The exoproteases regulation of P. larvae could be associated with QS. A promising mechanism of AFB control is to block QS mechanism with essential oils (EO. The aim of this study was to investigate the potential presence of QS signals in the regulation of P. larvae proteases and the effect of seven EOs on the exoproteases activity of P. larvae. From growth curves and evaluation of the presence of proteases by milk agar plates assay, it was observed protease activity during the late exponential phase of growth. Early production of protease activity (15 hours earlier than control was observed when a low density culture was incubated with late exponential spent medium (SM suggesting the presence of factor(s inducing this activity. SM was obtained by the ultrafiltration of P. larvae cultures on late growth phase and was free of proteases. Proteolytic activity was quantified on P. larvae cultures in presence of sublethal concentration of EO by azocasein method. The EOs, except S. chilensis EO, reduced significantly protease activity (more than 50%. We report for the first time evidence on the possible role of QS on P. larvae and the antiproteolytic activity of EOs (except for S. chilensis on exoproteases, an interesting therapeutic strategy to control AFB.

Allicin and derivates are cysteine protease inhibitors with antiparasitic activity.

Science.gov (United States)

Waag, Thilo; Gelhaus, Christoph; Rath, Jennifer; Stich, August; Leippe, Matthias; Schirmeister, Tanja

2010-09-15

Allicin and derivatives thereof inhibit the CAC1 cysteine proteases falcipain 2, rhodesain, cathepsin B and L in the low micromolar range. The structure-activity relationship revealed that only derivatives with primary carbon atom in vicinity to the thiosulfinate sulfur atom attacked by the active-site Cys residue are active against the target enzymes. Some compounds also show potent antiparasitic activity against Plasmodium falciparum and Trypanosoma brucei brucei. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

tolerant alkaline protease from Bacillus coagulans PSB

African Journals Online (AJOL)

oyaide

2013-05-22

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

Esterase and protease activities of Bacillus spp. from afitin, iru and ...

African Journals Online (AJOL)

The electrophoretic profiles of fermented African locust bean protein (ALBP), using strains presenting the highest protease activities in casein agar, were analyzed by SDS-PAGE to select strains with good ability to be used as starter cultures. All the Bacillus spp. tested showed esterase activity against tributyrin with high ...

Antiretroviral activity of protease inhibitors against Toxoplasma gondii

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Lianet Monzote

2013-02-01

Full Text Available The introduction of highly active antiretroviral therapy (HAART has caused a marked reduction in the occurrence and severity of parasitic infections, including the toxoplasmic encephalitis (TE. These changes have been attributed to the restoration of cell-mediated immunity. This study was developed to examine the activity of six antiretroviral protease inhibitors (API on Toxoplasma gondii tachyzoites. The six API showed anti-Toxoplasma activity, with IC50 value between 1.4 and 6.6 µg/mL. Further studies at the molecular level should be performed to clarify if the use of API could be beneficial or not for AIDS patients with TE.

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

KAUST Repository

Li, Zhao

2018-02-16

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

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

KAUST Repository

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

2018-01-01

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

Serum protease activity in chronic kidney disease patients: The GANI_MED renal cohort.

Science.gov (United States)

Wolke, Carmen; Teumer, Alexander; Endlich, Karlhans; Endlich, Nicole; Rettig, Rainer; Stracke, Sylvia; Fiene, Beate; Aymanns, Simone; Felix, Stephan B; Hannemann, Anke; Lendeckel, Uwe

2017-03-01

Serum or plasma proteases have been associated with various diseases including cancer, inflammation, or reno-cardiovascular diseases. We aimed to investigate whether the enzymatic activities of serum proteases are associated with the estimated glomerular filtration rate (eGFR) in patients with different stages of chronic kidney disease (CKD). Our study population comprised 268 participants of the "Greifswald Approach to Individualized Medicine" (GANI_MED) cohort. Enzymatic activity of aminopeptidase A, aminopeptidase B, alanyl (membrane) aminopeptidase, insulin-regulated aminopeptidase, puromycin-sensitive aminopeptidase, leucine aminopeptidase 3, prolyl-endopeptidase (PEP), dipeptidyl peptidase 4 (DPP4), angiotensin I-converting enzyme, and angiotensin I-converting enzyme 2 (ACE2) proteases was measured in serum. Linear regression of the respective protease was performed on kidney function adjusted for age and sex. Kidney function was modeled either by the continuous Modification of Diet in Renal Disease (MDRD)-based eGFR or dichotomized by eGFR < 15 mL/min/1.73 m 2 or <45 mL/min/1.73 m 2 , respectively. Results with a false discovery rate below 0.05 were deemed statistically significant. Among the 10 proteases investigated, only the activities of ACE2 and DPP4 were correlated with eGFR. Patients with lowest eGFR exhibited highest DPP4 and ACE2 activities. DPP4 and PEP were correlated with age, but all other serum protease activities showed no associations with age or sex. Our data indicate that ACE2 and DPP4 enzymatic activity are associated with the eGFR in patients with CKD. This finding distinguishes ACE2 and DPP4 from other serum peptidases analyzed and clearly indicates that further analyses are warranted to identify the precise role of these serum ectopeptidases in the pathogenesis of CKD and to fully elucidate underlying molecular mechanisms. Impact statement • Renal and cardiac diseases are very common and often occur concomitantly

Inhibition of proteases activity in intestine needs a sustainable acidic environment rather than a transient.

Science.gov (United States)

Xing, Chang; Xing, Jin-Feng; Ge, Zhi-Qiang

2017-10-01

α-Chymotrypsin (α-CT) and trypsin are important components of the enzymatic barrier. They could degrade the therapeutic proteins and peptides, inhibit their activity consequently, and thereby reduce their oral bioavailability. Acidic agents, as one type of indirect protease inhibitors, have shown proof of concept in clinical trials. We report here the inactivated proteases due to acid influence can be reactivated immediately by environmental pH recovery regardless of how long the inactivation last. To keep the inactivation time of proteases for 4-5 h, we designed and prepared a sustained-release tablet containing citric acid (CA) which can effectively reduce the pH below 5.0 and maintain it for 5 h in the dissolution-reaction medium. The activity of α-CT and trypsin was quantified by analyzing the residual amount of their respective substrates BTEE and TAME. More than 80% of the substrates were survived in 5.0 h of incubation, whereas the common tablet inhibited the proteases activity for only two hours in the same experimental medium. It indicates that the sustained-release tablet loaded with CA can efficiently inhibit the α-CT and trypsin activity longer than the common tablet. The results will be beneficial for designing and formulating the peroral administration of peptide and protein drugs.

Activity, specificity, and probe design for the smallpox virus protease K7L.

Science.gov (United States)

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

2012-11-16

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

Rheumatic Disease: Protease-Activated Receptor-2 in Synovial Joint Pathobiology

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Kendal McCulloch

2018-05-01

Full Text Available Protease-activated receptor-2 (PAR2 is one member of a small family of transmembrane, G-protein-coupled receptors. These receptors are activated via cleavage of their N terminus by serine proteases (e.g., tryptase, unveiling an N terminus tethered ligand which binds to the second extracellular loop of the receptor. Increasing evidence has emerged identifying key pathophysiological roles for PAR2 in both rheumatoid arthritis (RA and osteoarthritis (OA. Importantly, this includes both pro-inflammatory and destructive roles. For example, in murine models of RA, the associated synovitis, cartilage degradation, and subsequent bone erosion are all significantly reduced in the absence of PAR2. Similarly, in experimental models of OA, PAR2 disruption confers protection against cartilage degradation, subchondral bone osteosclerosis, and osteophyte formation. This review focuses on the role of PAR2 in rheumatic disease and its potential as an important therapeutic target for treating pain and joint degradation.

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

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Raheem Ullah

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

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

Science.gov (United States)

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

2016-01-01

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

Salmon trypsin stimulates the expression of interleukin-8 via protease-activated receptor-2

International Nuclear Information System (INIS)

Larsen, Anett K.; Seternes, Ole-Morten; Larsen, Merethe; Aasmoe, Lisbeth; Bang, Berit

2008-01-01

In this study, we focus on salmon trypsin as an activator of inflammatory responses in airway cells in vitro. The rationale behind the investigation is that salmon industry workers are exposed to aerosols containing enzymes, which are generated during industrial processing of the fish. Knowing that serine proteases such as trypsin are highly active mediators with diverse biological activities, the stimulation of nuclear factor-kappa B (NF-κB) and interleukin (IL)-8 and the role of protease-activated receptors (PAR) in inflammatory signal mediation were investigated. Protease-activated receptors are considered important under pathological situations in the human airways, and a thorough understanding of PAR-induced cellular events and their consequences in airway inflammation is necessary. Human airway epithelial cells (A549) were exposed to trypsin isolated from fish (Salmo salar), and we observed that purified salmon trypsin could generate secretion of IL-8 in a concentration-dependent manner. Furthermore, we demonstrate that PAR-2 activation by salmon trypsin is coupled to an induction of NF-κB-mediated transcription using a PAR-2 transfected HeLa cell model. Finally, we show that the release of IL-8 from A549 following stimulation with purified salmon trypsin is mediated through activation of PAR-2 using specific small interfering RNAs (siRNAs). The results presented suggest that salmon trypsin, via activation of PAR-2, might influence inflammation processes in the airways if inhaled in sufficient amounts

Extracellular proteases of Halobacillus blutaparonensis strain M9, a new moderately halophilic bacterium.

Science.gov (United States)

Santos, Anderson F; Valle, Roberta S; Pacheco, Clarissa A; Alvarez, Vanessa M; Seldin, Lucy; Santos, André L S

2013-12-01

Halophilic microorganisms are source of potential hydrolytic enzymes to be used in industrial and/or biotechnological processes. In the present study, we have investigated the ability of the moderately halophilic bacterium Halobacillus blutaparonensis (strain M9), a novel species described by our group, to release proteolytic enzymes. This bacterial strain abundantly proliferated in Luria-Bertani broth supplemented with 2.5% NaCl as well as secreted proteases to the extracellular environment. The production of proteases occurred in bacterial cells grown under different concentration of salt, ranging from 0.5% to 10% NaCl, in a similar way. The proteases secreted by H. blutaparonensis presented the following properties: (i) molecular masses ranging from 30 to 80 kDa, (ii) better hydrolytic activities under neutral-alkaline pH range, (iii) expression modulated according to the culture age, (iv) susceptibility to phenylmethylsulphonyl fluoride, classifying them as serine-type proteases, (v) specific cleavage over the chymotrypsin substrate, and (vi) enzymatic stability in the presence of salt (up to 20% NaCl) and organic solvents (e.g., ether, isooctane and cyclohexane). The proteases described herein are promising for industrial practices due to its haloalkaline properties.

PARTIAL PURIFICATION AND CHARACTERIZATION OF ALKALOPHILIC PROTEASE FROM PSEUDOMONAS AERUGINOSA

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R. Satheeskumar

2013-10-01

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

Characterization of a membrane-associated serine protease in Escherichia coli

International Nuclear Information System (INIS)

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

1987-01-01

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

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

DEFF Research Database (Denmark)

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

2013-01-01

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

Hepatitis C virus NS3/4A protease inhibits complement activation by cleaving complement component 4.

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Seiichi Mawatari

Full Text Available BACKGROUND: It has been hypothesized that persistent hepatitis C virus (HCV infection is mediated in part by viral proteins that abrogate the host immune response, including the complement system, but the precise mechanisms are not well understood. We investigated whether HCV proteins are involved in the fragmentation of complement component 4 (C4, composed of subunits C4α, C4β, and C4γ, and the role of HCV proteins in complement activation. METHODS: Human C4 was incubated with HCV nonstructural (NS 3/4A protease, core, or NS5. Samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then subjected to peptide sequencing. The activity of the classical complement pathway was examined using an erythrocyte hemolysis assay. The cleavage pattern of C4 in NS3/4A-expressing and HCV-infected cells, respectively, was also examined. RESULTS: HCV NS3/4A protease cleaved C4γ in a concentration-dependent manner, but viral core and NS5 did not. A specific inhibitor of NS3/4A protease reduced C4γ cleavage. NS3/4A protease-mediated cleavage of C4 inhibited classical pathway activation, which was abrogated by a NS3/4A protease inhibitor. In addition, co-transfection of cells with C4 and wild-type NS3/4A, but not a catalytic-site mutant of NS3/4A, produced cleaved C4γ fragments. Such C4 processing, with a concomitant reduction in levels of full-length C4γ, was also observed in HCV-infected cells expressing C4. CONCLUSIONS: C4 is a novel cellular substrate of the HCV NS3/4A protease. Understanding disturbances in the complement system mediated by NS3/4A protease may provide new insights into the mechanisms underlying persistent HCV infection.

Curcumin derivatives as HIV-1 protease inhibitors

Energy Technology Data Exchange (ETDEWEB)

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

1993-12-31

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

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

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Qing-Xiu Hu

2012-01-01

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

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

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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.

Insight into synergetic mechanisms of tetracycline and the selective serotonin reuptake inhibitor, sertraline, in a tetracycline-resistant strain of Escherichia coli

DEFF Research Database (Denmark)

Li, Lili; Kromann, Sofie; Olsen, John Elmerdahl

2017-01-01

further decreases pH, resulting in cell death. This study shows that sertraline interacts with tetracycline in a synergistic and AcrAB-TolC pump-independent manner. The combinational treatment was further shown to induce many changes in the global transcriptome, including altered tetA and tetR expression......Sertraline, an antidepressive drug, has been reported to inhibit general bacterial efflux pumps. In the present study, we report for the first time a synergistic effect of sertraline and tetracycline in a TetA-encoded tetracycline-resistant strain of Escherichia coli. Synergy between sertraline...... '1). RNA data suggest changes in respiration that is likely to decrease intracellular pH and thereby the proton-motive force, which provides the energy for the tetracycline efflux pump. Furthermore, sertraline and tetracycline may induce a change from oxidation to fermentation in the E.coli, which...

Cytomegalovirus protease targeted prodrug development.

Science.gov (United States)

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

2013-04-01

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

Tetracycline treatment of periodontal disease in the beagle dog

Energy Technology Data Exchange (ETDEWEB)

Jeffcoat, M.K.; Williams, R.C.; Kaplan, M.L.; Goldhaber, P.

1982-01-01

Bone-seeking radiopharmaceutical uptake (BSRU) was used to examine alveolar bone metabolism in a longitudinal study of tetracycline efficacy in beagle dogs. BSRU was measured in untreated control dogs and in beagles receiving either 250 mg or 500 mg oral tetracycline-HCl daily for 16 months. The rate of bone loss was determined for radiographs taken semiannually for a 6-month pretreatment period and for a 24-month treatment period. Measurements of BSRU obtained at month 16 of treatment were correlated with rates of bone loss determined radiographically in an attempt to determine whether BSRU was indicative of the subsequent rate of bone loss. A reduced rate of alveolar bone loss was found in the 500 mg tetracycline group at month 16 of the study relative to the untreated controls which was consistent with the decreased bone-seeking radiopharmaceutical uptake observed in this group. A significantly increased BSRU (p<.0001) was found in the 250 mg tetracycline group at month 16 of study relative to the untreated and 500 mg tetracycline groups. A rapid increase in the rate of bone loss in the 250 mg tetracycline group which was not detectable prior to 16 months of treatment became evident radiographically by 24 months. Thus, increased BSRU in the 250 mg tetracycline group appeared to detect the loss of the effect of tetracycline (escape phenomenon). 13 references, 4 figures.

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

Science.gov (United States)

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

2017-10-01

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

The LasB Elastase of Pseudomonas aeruginosa Acts in Concert with Alkaline Protease AprA To Prevent Flagellin-Mediated Immune Recognition.

Science.gov (United States)

Casilag, Fiordiligie; Lorenz, Anne; Krueger, Jonas; Klawonn, Frank; Weiss, Siegfried; Häussler, Susanne

2016-01-01

The opportunistic pathogen Pseudomonas aeruginosa is capable of establishing severe and persistent infections in various eukaryotic hosts. It encodes a wide array of virulence factors and employs several strategies to evade immune detection. In the present study, we screened the Harvard Medical School transposon mutant library of P. aeruginosa PA14 for bacterial factors that modulate interleukin-8 responses in A549 human airway epithelial cells. We found that in addition to the previously identified alkaline protease AprA, the elastase LasB is capable of degrading exogenous flagellin under calcium-replete conditions and prevents flagellin-mediated immune recognition. Our results indicate that the production of two proteases with anti-flagellin activity provides a failsafe mechanism for P. aeruginosa to ensure the maintenance of protease-dependent immune-modulating functions. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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

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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.

Host regulation of lysogenic decision in bacteriophage lambda: transmembrane modulation of FtsH (HflB), the cII degrading protease, by HflKC (HflA).

Science.gov (United States)

Kihara, A; Akiyama, Y; Ito, K

1997-05-27

The cII gene product of bacteriophage lambda is unstable and required for the establishment of lysogenization. Its intracellular amount is important for the decision between lytic growth and lysogenization. Two genetic loci of Escherichia coli are crucial for these commitments of infecting lambda genome. One of them, hflA encodes the HflKC membrane protein complex, which has been believed to be a protease degrading the cII protein. However, both its absence and overproduction stabilized cII in vivo and the proposed serine protease-like sequence motif in HflC was dispensable for the lysogenization control. Moreover, the HflKC protein was found to reside on the periplasmic side of the plasma membrane. In contrast, the other host gene, ftsH (hflB) encoding an integral membrane ATPase/protease, is positively required for degradation of cII, since loss of its function stabilized cII and its overexpression accelerated the cII degradation. In vitro, purified FtsH catalyzed ATP-dependent proteolysis of cII and HflKC antagonized the FtsH action. These results, together with our previous finding that FtsH and HflKC form a complex, suggest that FtsH is the cII degrading protease and HflKC is a modulator of the FtsH function. We propose that this transmembrane modulation differentiates the FtsH actions to different substrate proteins such as the membrane-bound SecY protein and the cytosolic cII protein. This study necessitates a revision of the prevailing view about the host control over lambda lysogenic decision.

Bacterial Resistance to the Tetracyclines and Antimicrobial ...

African Journals Online (AJOL)

Optimizing of tetracycline antibiotics dosing and duration in human and animal healthcare and food production might help minimize the emergence of resistance in some situations. New approaches to antimicrobial chemotherapy are needed if we are to survive the increasing rates of tetracycline antibiotic resistance ...

Engineered Toxins “Zymoxins” Are Activated by the HCV NS3 Protease by Removal of an Inhibitory Protein Domain

Science.gov (United States)

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

Vitamin D modulates tissue factor and protease-activated receptor 2 expression in vascular smooth muscle cells.

Science.gov (United States)

Martinez-Moreno, Julio M; Herencia, Carmen; Montes de Oca, Addy; Muñoz-Castañeda, Juan R; Rodríguez-Ortiz, M Encarnación; Díaz-Tocados, Juan M; Peralbo-Santaella, Esther; Camargo, Antonio; Canalejo, Antonio; Rodriguez, Mariano; Velasco-Gimena, Francisco; Almaden, Yolanda

2016-03-01

Clinical and epidemiologic studies reveal an association between vitamin D deficiency and increased risk of cardiovascular disease. Because vascular smooth muscle cell (VSMC)-derived tissue factor (TF) is suggested to be critical for arterial thrombosis, we investigated whether the vitamin D molecules calcitriol and paricalcitol could reduce the expression of TF induced by the proinflammatory cytokine TNF-α in human aortic VSMCs. We found that, compared with controls, incubation with TNF-α increased TF expression and procoagulant activity in a NF-κB-dependent manner, as deduced from the increased nuclear translocation of nuclear factor κ-light-chain-enhancer of activated B cells protein 65 (p65-NF-κB) and direct interaction of NF-κB to the TF promoter. This was accompanied by the up-regulation of TF signaling mediator protease-activated receptor 2 (PAR-2) expression and by the down-regulation of vitamin D receptor expression in a miR-346-dependent way. However, addition of calcitriol or paricalcitol blunted the TNF-α-induced TF expression and activity (2.01 ± 0.24 and 1.32 ± 0.14 vs. 3.02 ± 0.39 pmol/mg protein, P < 0.05), which was associated with down-regulation of NF-κB signaling and PAR-2 expression, as well as with restored levels of vitamin D receptor and enhanced expression of TF pathway inhibitor. Our data suggest that inflammation promotes a prothrombotic state through the up-regulation of TF function in VSMCs and that the beneficial cardiovascular effects of vitamin D may be partially due to decreases in TF expression and its activity in VSMCs. © FASEB.

Studies on activity, distribution, and zymogram of protease, α-amylase, and lipase in the paddlefish Polyodon spathula.

Science.gov (United States)

Ji, H; Sun, H T; Xiong, D M

2012-06-01

A series of biochemical determination and electrophoretic observations have been conducted to analyze the activities and characteristics of protease, α-amylase, and lipase of paddlefish Polyodon spathula. The results obtained have been compared with those of bighead carp (Aristichthys nobilis) and hybrid sturgeon (Huso dauricus ♀ × Acipenser schrenki Brandt ♂), in order to increase available knowledge of the physiological characteristics of this sturgeon species and to gain information with regard to its nutrition. Further, a comparative study of enzymatic activity, distribution, and characterization between commercial feed-reared paddlefish (CG) and natural live food-reared (NG) paddlefish was conducted. Results showed that higher proteolytic activity was observed in the pH range 2.5-3.0 and at a pH of 7.0 for paddlefish. Levels of acid protease activity of paddlefish were similar to that of hybrid sturgeon, and significantly higher than that of bighead carp. The inhibition assay of paddlefish showed that the rate of inhibition of tosyl-phenylalanine chloromethyl ketone was approximately 2.6-fold that of tosyl-lysine chloromethyl ketone. There was no significant difference observed for acid protease activity between PG and CG groups, whereas the activity of alkaline protease, α-amylase, and lipase in the PG group were significantly lower than those in the CG group. The substrate sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis further showed that there were certain types of enzymes, especially α-amylase, with similar molecular mass in the paddlefish and hybrid sturgeon. It can be inferred that acid digestion was main mechanism for protein hydrolysis in paddlefish, as reported for other fishes with a stomach. This indicates that the paddlefish requires higher alkaline protease, α-amylase, and lipase activity to digest natural live food.

Organic acids enhance bioavailability of tetracycline in water to Escherichia coli for uptake and expression of antibiotic resistance.

Science.gov (United States)

Zhang, Yingjie; Boyd, Stephen A; Teppen, Brian J; Tiedje, James M; Li, Hui

2014-11-15

Tetracyclines are a large class of antimicrobials used most extensively in livestock feeding operations. A large portion of tetracyclines administered to livestock is excreted in manure and urine which is collected in waste lagoons. Subsequent land application of these wastes introduces tetracyclines into the soil environment, where they could exert selective pressure for the development of antibiotic resistance genes in bacteria. Tetracyclines form metal-complexes in natural waters, which could reduce their bioavailability for bacterial uptake. We hypothesized that many naturally-occurring organic acids could effectively compete with tetracyclines as ligands for metal cations, hence altering the bioavailability of tetracyclines to bacteria in a manner that could enhance the selective pressure. In this study, we investigated the influence of acetic acid, succinic acid, malonic acid, oxalic acid and citric acid on tetracycline uptake from water by Escherichia coli bioreporter construct containing a tetracycline resistance gene which induces the emission of green fluorescence when activated. The presence of the added organic acid ligands altered tetracycline speciation in a manner that enhanced tetracycline uptake by E. coli. Increased bacterial uptake of tetracycline and concomitant enhanced antibiotic resistance response were quantified, and shown to be positively related to the degree of organic acid ligand complexation of metal cations in the order of citric acid > oxalic acid > malonic acid > succinic acid > acetic acid. The magnitude of the bioresponse increased with increasing aqueous organic acid concentration. Apparent positive relation between intracellular tetracycline concentration and zwitterionic tetracycline species in aqueous solution indicates that (net) neutral tetracycline is the species which most readily enters E. coli cells. Understanding how naturally-occurring organic acid ligands affect tetracycline speciation in solution, and how speciation

Fluorescently labeled inhibitors detect localized serine protease activities in Drosophila melanogaster pole cells, embryos, and ovarian egg chambers

DEFF Research Database (Denmark)

Jakobsen, Rasmus Kragh; Ono, S.; Powers, J. C.

2005-01-01

processes that they mediate. Until only recently, the tools to conveniently address the question of where and when serine proteases are active within complex tissues have been lacking. In order to detect spatially restricted serine protease activities in Drosophila embryos and ovaries we introduce...... activity localized to the oocyte-somatic follicle cell interface of the developing egg chamber. Our results suggest that this technique holds promise to identify new spatially restricted activities in adult Drosophila tissues and developing embryos....

Deletion of protease-activated receptor 2 prolongs survival of scrapie-inoculated mice

Czech Academy of Sciences Publication Activity Database

Matěj, R.; Olejár, Tomáš; Janoušková, O.; Holada, K.

2012-01-01

Roč. 93, č. 9 (2012), s. 2057-2061 ISSN 0022-1317 Institutional support: RVO:67985823 Keywords : protease-activated receptor (PAR2) * scrapie * neurodegenerative disorders Subject RIV: FN - Epidemiology, Contagious Diseases ; Clinical Immunology Impact factor: 3.127, year: 2012

Clp Protease and OR Directly Control the Proteostasis of Phytoene Synthase, the Crucial Enzyme for Carotenoid Biosynthesis in Arabidopsis.

Science.gov (United States)

Welsch, Ralf; Zhou, Xiangjun; Yuan, Hui; Álvarez, Daniel; Sun, Tianhu; Schlossarek, Dennis; Yang, Yong; Shen, Guoxin; Zhang, Hong; Rodriguez-Concepcion, Manuel; Thannhauser, Theodore W; Li, Li

2018-01-08

Phytoene synthase (PSY) is the crucial plastidial enzyme in the carotenoid biosynthetic pathway. However, its post-translational regulation remains elusive. Likewise, Clp protease constitutes a central part of the plastid protease network, but its substrates for degradation are not well known. In this study, we report that PSY is a substrate of the Clp protease. PSY was uncovered to physically interact with various Clp protease subunits (i.e., ClpS1, ClpC1, and ClpD). High levels of PSY and several other carotenogenic enzyme proteins overaccumulate in the clpc1, clpp4, and clpr1-2 mutants. The overaccumulated PSY was found to be partially enzymatically active. Impairment of Clp activity in clpc1 results in a reduced rate of PSY protein turnover, further supporting the role of Clp protease in degrading PSY protein. On the other hand, the ORANGE (OR) protein, a major post-translational regulator of PSY with holdase chaperone activity, enhances PSY protein stability and increases the enzymatically active proportion of PSY in clpc1, counterbalancing Clp-mediated proteolysis in maintaining PSY protein homeostasis. Collectively, these findings provide novel insights into the quality control of plastid-localized proteins and establish a hitherto unidentified post-translational regulatory mechanism of carotenogenic enzymes in modulating carotenoid biosynthesis in plants. Copyright © 2017 The Author. Published by Elsevier Inc. All rights reserved.

Effect of tetracycline residues in pig manure slurry on tetracycline-resistant bacteria and resistance gene tet(M) in soil microcosms

DEFF Research Database (Denmark)

Agersø, Yvonne; Wulff, Gitte; Vaclavik, Elvira

2006-01-01

and oxytetracycline were almost stable through out the experimental period, but the tetracycline concentrations had no effect on prevalence of tetracycline-resistant bacteria. The presented microcosm approach simulated natural farmland conditions well and supported results from previous field studies. (c) 2006...

Tetracycline Reduces Kidney Damage Induced by Loxosceles Spider Venom

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Cinthya Kimori Okamoto

2017-03-01

Full Text Available Envenomation by Loxosceles spider can result in two clinical manifestations: cutaneous and systemic loxoscelism, the latter of which includes renal failure. Although incidence of renal failure is low, it is the main cause of death, occurring mainly in children. The sphingomyelinase D (SMase D is the main component in Loxosceles spider venom responsible for local and systemic manifestations. This study aimed to investigate the toxicity of L. intermedia venom and SMase D on kidney cells, using both In vitro and in vivo models, and the possible involvement of endogenous metalloproteinases (MMP. Results demonstrated that venom and SMase D are able to cause death of human kidney cells by apoptosis, concomitant with activation and secretion of extracellular matrix metalloproteases, MMP-2 and MMP-9. Furthermore, cell death and MMP synthesis and secretion can be prevented by tetracycline. In a mouse model of systemic loxoscelism, Loxosceles venom-induced kidney failure was observed, which was abrogated by administration of tetracycline. These results indicate that MMPs may play an important role in Loxosceles venom-induced kidney injury and that tetracycline administration may be useful in the treatment of human systemic loxoscelism.

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

Science.gov (United States)

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

2017-06-01

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

Biosolid-borne tetracyclines and sulfonamides in plants.

Science.gov (United States)

Mathews, Shiny; Reinhold, Dawn

2013-07-01

Tetracyclines and sulfonamides used in human and animal medicine are released to terrestrial ecosystems from wastewater treatment plants or by direct manure application. The interactions between plants and these antibiotics are numerous and complex, including uptake and accumulation, phytometabolism, toxicity responses, and degradation in the rhizosphere. Uptake and accumulation of antibiotics have been studied in plants such as wheat, maize, potato, vegetables, and ornamentals. Once accumulated in plant tissue, organic contaminants can be metabolized through a sequential process of transformation, conjugation through glycosylation and glutathione pathways, and ultimately sequestration into plant tissue. While studies have yet to fully elucidate the phytometabolism of tetracyclines and sulfonamides, an in-depth review of plant and mammalian studies suggest multiple potential transformation and conjugation pathways for tetracyclines and sulfonamides. The presence of contaminants in the vicinity or within the plants can elicit stress responses and defense mechanisms that can help tolerate the negative effects of contaminants. Antibiotics can change microbial communities and enzyme activity in the rhizosphere, potentially inducing microbial antibiotic resistance. On the other hand, the interaction of microbes and root exudates on pharmaceuticals in the rhizosphere can result in degradation of the parent molecule to less toxic compounds. To fully characterize the environmental impacts of increased antibiotic use in human medicine and animal production, further research is essential to understand the effects of different antibiotics on plant physiology and productivity, uptake, translocation, and phytometabolism of antibiotics, and the role of antibiotics in the rhizosphere.

Interaction of protein C inhibitor with the type II transmembrane serine protease enteropeptidase.

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Thomas A Prohaska

Full Text Available The serine protease inhibitor protein C inhibitor (PCI is expressed in many human tissues and exhibits broad protease reactivity. PCI binds glycosaminoglycans and certain phospholipids, which modulate its inhibitory activity. Enteropeptidase (EP is a type II transmembrane serine protease mainly found on the brush border membrane of epithelial cells in the duodenum, where it activates trypsinogen to initiate the digestion of food proteins. Some active EP is also present in duodenal fluid and has been made responsible for causing pancreatitis in case of duodeno-pancreatic reflux. Together with its substrate trypsinogen, EP is furthermore present in the epidermis and in some cancer cells. In this report, we show that PCI inhibited EP with an apparent 2nd order rate constant of 4.48 × 10(4 M(-1 s(-1. Low molecular weight (LMWH and unfractionated heparin (UFH slightly reduced the inhibitory effect of PCI. The SI (stoichiometry of inhibition value for the inhibition of EP by PCI was 10.8 in the absence and 17.9 in the presence of UFH (10 U/ml. By inhibiting trypsin, chymotrypsin, and additionally EP, PCI might play a role in the protection of the pancreas from autodigestion. Furthermore the interaction of PCI with EP may influence the regulation of epithelial differentiation.

Skeletal muscle protease activities in the early growth and development of wild Atlantic salmon (Salmo salar L.).

Science.gov (United States)

Lysenko, Liudmila A; Kantserova, Nadezda P; Kaivarainen, Elena I; Krupnova, Marina Yu; Nemova, Nina N

2017-09-01

Growth-related dynamics of intracellular protease activities in four year classes of the Atlantic salmon (Salmo salar L. 1758) parr and smolts inhabiting salmon rivers of northwestern Russia (the White Sea basin) were studied. Cathepsin B, cathepsin D, proteasome, and calpain activities in the skeletal muscles of salmon were assessed to investigate their relative contribution to the total protein degradation as well as to young fish growth process. It was confirmed that calpain activity dominates in salmon muscles while proteasome plays a minor role, in contrast to terrestrial vertebrates. Calpain and proteasome activities were maximal at the early post-larval stage (in parrs 0+) and declined with age (parrs 1+ through 2+) dropping to the lowest level in salmon smolts. Annual growth increments and proteolytic activities of calpains and proteasome in the muscles of salmon juveniles changed with age in an orchestrated manner, while lysosomal cathepsin activities increased with age. Comparing protease activities and growth increments in salmon parr and smolts we suggested that the partial suppression of the protein degradation could be a mechanism stimulating efficient growth in smoltifying salmon. Growth and smoltification-related dynamics of protease activities was quite similar in salmon populations from studied spawning rivers, such as Varzuga and Indera; however, some habitat-related differences were observed. Growth increments and protease activities varied in salmon parr 0+ (but not on later ages) inhabiting either main rivers or small tributaries apparently due to habitat difference on the resources for fish growth. Copyright © 2017 Elsevier Inc. All rights reserved.

Removal of tetracycline from contaminated water by Moringa oleifera seed preparations.

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Santos, Andréa F S; Matos, Maria; Sousa, Ângela; Costa, Cátia; Nogueira, Regina; Teixeira, José A; Paiva, Patrícia M G; Parpot, Pier; Coelho, Luana C B B; Brito, António G

2016-01-01

The aim of this study was to evaluate tetracycline antibiotic (TA) removal from contaminated water by Moringa oleifera seed preparations. The composition of synthetic water approximate river natural contaminated water and TA simulated its presence as an emerging pollutant. Interactions between TA and protein preparations (extract; fraction and lectin) were also evaluated. TA was determined by solid-phase extraction followed by high-performance liquid chromatography-mass spectrometry. Moringa extract and flour removed TA from water. The extract removed TA in all concentrations, and better removal (40%) was obtained with 40 mg L(-1); seed flour (particles  5 mm (0.50 g L(-1)) removed 55% of antibiotic. Interactions between TA and seed preparations were assayed by haemagglutinating activity (HA). Specific HA (SHA) of extract (pH 7) was abolished with tetracycline (5 mg L(-1)); fraction (75%) and lectin HA (97%) were inhibited with TA. Extract SHA decreased by 75% at pH 8. Zeta potential (ZP) of extract 700 mg L(-1) and tetracycline 50 mg L(-1) , pH range 5-8, showed different results. Extract ZP was more negative (-10.73 to -16.00 mV) than tetracycline ZP (-0.27 to -20.15 mV); ZP difference was greater in pH 8. The focus of this study was achieved since Moringa preparations removed TA from water and compounds interacting with tetracycline involved at least lectin-binding sites. This is a natural process, which do not promote environmental damage.

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

Science.gov (United States)

Lerdsirisuk, Pradith; Maicheen, Chirattikan; Ungwitayatorn, Jiraporn

2014-12-01

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

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

DEFF Research Database (Denmark)

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

2016-01-01

stringent sequence specificity and enhanced activity. Like other proteases, activity of the human rhinovirus 3C protease can be affected in part by the buffer components and additives that are generally employed for purification and stabilization of proteins, hence, necessitate their removal by tedious...

Insecticide resistance and intracellular proteases.

Science.gov (United States)

Wilkins, Richard M

2017-12-01

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

Modulating effects of the protease inhibitor Antipain on x-ray induced transformations

International Nuclear Information System (INIS)

Borek, C.; Miller, R.C.

1979-01-01

Protease inhibitors have been shown to inhibit the expression of mutations in bacteria and to inhibit the tumor-promoting effect of phorbol esters in mice. We have investigated the effect of the protease inhibitor Antipain on cell transformation by x-irradiation in two in vitro systems; namely short-term cultures of freshly explanted hamster embryo cells and in the 10T1/2 cell line derived and cloned from C3H mouse embryo

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior.

Science.gov (United States)

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H; Muyldermans, Serge; Declerck, Paul J; Huang, Mingdong; Andreasen, Peter A; Ngo, Jacky Chi Ki

2016-07-15

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30-40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

A Camelid-derived Antibody Fragment Targeting the Active Site of a Serine Protease Balances between Inhibitor and Substrate Behavior*

Science.gov (United States)

Kromann-Hansen, Tobias; Oldenburg, Emil; Yung, Kristen Wing Yu; Ghassabeh, Gholamreza H.; Muyldermans, Serge; Declerck, Paul J.; Huang, Mingdong; Andreasen, Peter A.; Ngo, Jacky Chi Ki

2016-01-01

A peptide segment that binds the active site of a serine protease in a substrate-like manner may behave like an inhibitor or a substrate. However, there is sparse information on which factors determine the behavior a particular peptide segment will exhibit. Here, we describe the first x-ray crystal structure of a nanobody in complex with a serine protease. The nanobody displays a new type of interaction between an antibody and a serine protease as it inserts its complementary determining region-H3 loop into the active site of the protease in a substrate-like manner. The unique binding mechanism causes the nanobody to behave as a strong inhibitor as well as a poor substrate. Intriguingly, its substrate behavior is incomplete, as 30–40% of the nanobody remained intact and inhibitory after prolonged incubation with the protease. Biochemical analysis reveals that an intra-loop interaction network within the complementary determining region-H3 of the nanobody balances its inhibitor versus substrate behavior. Collectively, our results unveil molecular factors, which may be a general mechanism to determine the substrate versus inhibitor behavior of other protease inhibitors. PMID:27226628

Effects of cysteine on growth, protease production, and catalase activity of Pseudomonas fluorescens.

OpenAIRE

Himelbloom, B H; Hassan, H M

1986-01-01

Cysteine inhibits growth of and protease production by Pseudomonas fluorescens NC3. Catalase activity in P. fluorescens NC3 was increased by cysteine. The addition of exogenous hydrogen peroxide did not increase catalase activity, thus suggesting a role for the endogenous generation of hydrogen peroxide via the autoxidation of cysteine.

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

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Sato A

2015-04-01

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

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

Science.gov (United States)

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

2017-08-01

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

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

OpenAIRE

1984-01-01

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

Radiation deamination of tetracycline

International Nuclear Information System (INIS)

Kuduk-Jaworska, J.

1980-01-01

The fundamental product of tetracycline hydrochlorine gamma radiolysis was separated in its solid state. From the results of spectroscopic studies it has been established that it is des-N,N-dimethylaminotetracycline. (author)

Factor VII-activating protease : Unraveling the release and regulation of dead cell nuclear damps

NARCIS (Netherlands)

Marsman, G.

2017-01-01

Upon inflammation, uncleared dying cells are an important source of pro-inflammatory damage-associated molecular patterns (DAMPs). Major DAMPs are histones and double-stranded DNA, which together form chromatin. Factor VII-activating protease (FSAP) is activated upon contact with dead cells, and its

Supermarket Proteases.

Science.gov (United States)

Hagar, William G.; Bullerwell, Lornie D.

2003-01-01

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

Multifunctional Mitochondrial AAA Proteases.

Science.gov (United States)

Glynn, Steven E

2017-01-01

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

Effect of Seed Proteinaceous Inhibitors on Digestive Protease Activity of Tomato Leaf Miner Tuta absoluta (Meyrick (Lep.: Gelechiidae

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M. University of Tehran

2017-01-01

Full Text Available Introduction: Tomato leaf miner Tuta absoluta Meyrick (Lep.: Gelechiidae is one of the most important pest of tomato around the world which have been introduced to Iran in recent years. Pesticides can usually reduce the damage caused by the pest, however the extensive use of pesticides substantially increase production costs and lead to development of insecticide resistance. Thus, pest resistance and also adverse effects of pesticides on the human health and the environment make search for alternative control method/s inevitable. The aim of this study was to examine the effect of proteinaceous extracts of Datura (Datura stramonium L., Bean (Phaseolus vulgaris L., Wild Oat (Avena fatua L., Broad bean (Vicia faba L., Amaranth (Amaranthus retroflexus L., Mung bean (Vigna radiate L., Pea (Pisum sativum L., Canola (Brassica napus L., Triticale (Triticosecale wittmack and Sivand, Aflak and Alborz wheat (Triticum aestivum L. cultivars seeds on protease activity of tomato leaf miner. Also in this study optimum pH for protease activity and effects of pH on inhibitory activity of extracts were determined. Materials and Methods: For this work fourth instar larvae of Tuta absoluta was used for enzyme extraction. Plant proteinaceous extracts were obtained using 0.1 M NaCl. For protease activity assay 10 μl enzyme extract and 50 μl substrate solution (Azocasein 2% were mixed with 40 μl of the 20 mM Glycine-NaOH buffer at pH 10.0 After 60 min incubation, 100 μl of 30% trichloroacetic acid (TCA was added to the reaction mixture, and kept at 4 °C for 30 min, followed by centrifugation at 15,000gfor 15 min to precipitate non-hydrolysis substrate. 100 μl of 1 M NaOH was added to 100μl supernatant and the absorbance at 405 nm was measured. To determine the effect of the seed proteinaceous extracts on the protease activities, enzymes were pre-incubated with each seed extracts for 30 min at 35 °C followed by determination of the residual enzyme activity as

Relation between tetR and tetA expression in tetracycline resistant Escherichia coli

DEFF Research Database (Denmark)

Møller, Thea S. B.; Overgaard, Martin; Nielsen, Søren S.

2016-01-01

Background: Tetracyclines are among the most used antibiotics in livestock worldwide. Resistance is widely disseminated in Escherichia coli, where it is generally mediated by tetracycline efflux pumps, such as TetA. Expression of tetracycline efflux pumps is tightly controlled by the repressor Tet......R, which has been shown to be tetracycline-responsive at sub-MIC tetracycline concentrations. The objective of this study was to investigate the effects of increasing tetracycline concentrations on the growth of TetA-producing E. coli, and to determine how expression of tetA and tetR related to each other...... in different growth phases in the presence of tetracycline. Results: A tetracycline resistant E. coli strain containing tetA and tetR on the chromosome was constructed and cultured in the presence of increasing concentrations of tetracycline. Expression of tetR and tetA was measured at four time points...

Tetracyclines and tetracycline resistance in agricultural soils: microcosm and field studies.

NARCIS (Netherlands)

Schmitt, Heike; Stoob, Krispin; Hamscher, Gerd; Smit, Eric; Seinen, Willem

2006-01-01

The influence of the use of antibiotics on the prevalence of resistance genes in the environment is still poorly understood. We studied the diversity of tetracycline and sulfonamide resistance genes as influenced by fertilization with pig manure in soil microcosms and at two field locations. Manure

Effects of protease and non-starch polysaccharide enzyme on performance, digestive function, activity and gene expression of endogenous enzyme of broilers.

Directory of Open Access Journals (Sweden)

Lin Yuan

Full Text Available Three hundred one-day-old male broiler chickens (Ross-308 were fed corn-soybean basal diets containing non-starch polysaccharide (NSP enzyme and different levels of acid protease from 1 to 42 days of age to investigate the effects of exogenous enzymes on growth performance, digestive function, activity of endogenous digestive enzymes in the pancreas and mRNA expression of pancreatic digestive enzymes. For days 1-42, compared to the control chickens, average daily feed intake (ADFI and average daily gain (ADG were significantly enhanced by the addition of NSP enzyme in combination with protease supplementation at 40 or 80 mg/kg (p<0.05. Feed-to-gain ratio (FGR was significantly improved by supplementation with NSP enzymes or NSP enzyme combined with 40 or 80 mg/kg protease compared to the control diet (p<0.05. Apparent digestibility of crude protein (ADCP was significantly enhanced by the addition of NSP enzyme or NSP enzyme combined with 40 or 80 mg/kg protease (p<0.05. Cholecystokinin (CCK level in serum was reduced by 31.39% with NSP enzyme combined with protease supplementation at 160 mg/kg (p<0.05, but the CCK level in serum was increased by 26.51% with NSP enzyme supplementation alone. After 21 days, supplementation with NSP enzyme and NSP enzyme combined with 40 or 80 mg/kg protease increased the activity of pancreatic trypsin by 74.13%, 70.66% and 42.59% (p<0.05, respectively. After 42 days, supplementation with NSP enzyme and NSP enzyme combined with 40 mg/kg protease increased the activity of pancreatic trypsin by 32.45% and 27.41%, respectively (p<0.05. However, supplementation with NSP enzyme and 80 or 160 mg/kg protease decreased the activity of pancreatic trypsin by 10.75% and 25.88%, respectively (p<0.05. The activities of pancreatic lipase and amylase were significantly higher in treated animals than they were in the control group (p<0.05. Supplementation with NSP enzyme, NSP enzyme combined with 40 or 80 mg/kg protease increased

In vitro assay for the anti-Brucella activity of medicinal plants against tetracycline-resistant Brucella melitensis.

Science.gov (United States)

Motamedi, Hossein; Darabpour, Esmaeil; Gholipour, Mahnaz; Seyyed Nejad, Seyyed Mansour

2010-07-01

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50-400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria decumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study.

In vitro assay for the anti-brucella activity of medicinal plants against tetracycline-resistant Brucella melitensis *

Science.gov (United States)

Motamedi, Hossein; Darabpour, Esmaeil; Gholipour, Mahnaz; Seyyed Nejad, Seyyed Mansour

2010-01-01

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50–400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria decumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study. PMID:20593515

Continuous-flow chemiluminometric determination of some tetracyclines

International Nuclear Information System (INIS)

Syropoulos, A.B.; Calokerinos, A.C.

1991-01-01

Chemiluminescence is found to be generated by action of lucigenin or hexacyanoferrate(III) on tetracyclines. The reaction with lucigenin exhibits chemiluminescence after alkaline degradation of tetracyclines to the corresponding iso derivatives. The reaction with hexacyanoferrate (III) occurs after acidic degradation of tetracyclines to corresponding anhydro derivatives. The chemiluminescence reaction takes place in alkaline medium, and allows the development of a continuous-flow method for the determination of 1.00-10.0 μgml -1 oxytetracycline and doxycycline. When applied to commercial formulations, the procedure was relatively free from interferences from common excipients. The results obtained for the assay of dosage forms compared well with those obtained by the official methods and demonstrated good accuracy and precision. (author). 32 refs.; 5 figs.; 6 tabs

Continuous-flow chemiluminometric determination of some tetracyclines

Energy Technology Data Exchange (ETDEWEB)

Syropoulos, A B; Calokerinos, A C [University of Athens (greece). Laboratory of Analytical Chemistry

1991-12-24

Chemiluminescence is found to be generated by action of lucigenin or hexacyanoferrate(III) on tetracyclines. The reaction with lucigenin exhibits chemiluminescence after alkaline degradation of tetracyclines to the corresponding iso derivatives. The reaction with hexacyanoferrate (III) occurs after acidic degradation of tetracyclines to corresponding anhydro derivatives. The chemiluminescence reaction takes place in alkaline medium, and allows the development of a continuous-flow method for the determination of 1.00-10.0 {mu}gml{sup -1} oxytetracycline and doxycycline. When applied to commercial formulations, the procedure was relatively free from interferences from common excipients. The results obtained for the assay of dosage forms compared well with those obtained by the official methods and demonstrated good accuracy and precision. (author). 32 refs.; 5 figs.; 6 tabs.

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

OpenAIRE

Naiola, Elidar; Widhyastuti, Nunuk

2002-01-01

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

Syzygium jambos Displayed Antibacterial and Antibiotic-Modulating Activities against Resistant Phenotypes

Directory of Open Access Journals (Sweden)

Brice E. N. Wamba

2018-01-01

Full Text Available The present study was designed to evaluate the antibacterial activities of methanol extracts of bark and leaves of Syzygium jambos, as well as their synergistic effects with selected antibiotics against drug-resistant Gram-positive and Gram-negative bacteria. The crude extracts were subjected to qualitative phytochemical screening; broth microdilution method was used for antibacterial assays. Phytochemical studies indicate that leaves and bark extracts contained polyphenols, anthraquinones, tannins, and steroids. Extract of the leaves was active against all the 26 strains of Staphylococcus aureus and all the 21 strains of Gram-negative bacteria tested, within the minimum inhibitory concentration (MIC range of 32–512 μg/mL. The lowest MIC value of 32 μg/mL was obtained with extract of the leaves against Staphylococcus aureus MRSA9 strain. In Gram-negative bacteria, the lowest MIC value of 64 μg/mL was also obtained against Enterobacter aerogenes EA294 and Klebsiella pneumoniae K24 strains. Against S. aureus strains, antibiotic-modulating activity of extracts at MIC/2 towards more than 70% of the tested strains was obtained when leaves and bark extracts were tested in association with chloramphenicol (CHL. This was also the case when leaves extract was combined with CHL, kanamycin (KAN, tetracycline (TET, and erythromycin (ERY and when bark extract was combined with ciprofloxacin (CIP, TET, and ERY against Gram-negative bacteria. In conclusion, this study demonstrated that Syzygium jambos has antibacterial and antibiotic-modulating activities.

Studies on screening, isolation and purification of a fibrinolytic protease from an isolate (VK12) of Ganoderma lucidum and evaluation of its antithrombotic activity.

Science.gov (United States)

Kumaran, Sekar; Palani, Perumal; Nishanthi, Ramasami; Kaviyarasan, Venkatesan

2011-01-01

Antithrombotic activity of a protease purified from a medicinal mushroom, Ganoderma lucidum, has been evaluated platelet aggregation in vitro and pulmonary thrombosis in vivo. The purified protease exhibited concentration dependent inhibitory effects on platelet aggregation induced by ADP (adenosine diphosphate), with an IC(50) value of 2.4 mg/mL. The purified protease protected mice against thrombotic death or paralysis induced by collagen and epinephrine in a dose dependent manner when administered orally. It produced a significant inhibition of thrombotic death or paralysis at 60 µg/kg body weight, while aspirin produced a significant inhibition of thrombosis at 10-20 mg/kg body weight. The purified protease also has showed fibrinolytic activity and alters coagulation parameters such as activated partial thromboplastin time (APTT), and thrombin time (TT) in rat platelet. These results suggested that the antithrombotic activity of Ganoderma lucidum protease might be due to antiplatelet activity rather than anticoagulation activity.

Ligand Binding Domain Protein in Tetracycline-Inducible Expression

African Journals Online (AJOL)

Purpose: To investigate tetracycline-inducible expression system for producing clinically usable, highquality liver X receptor ligand-binding domain recombinant protein. Methods: In this study, we have expressed and purified the recombinant liver X receptor β-ligand binding domain proteins in E. coli using a tetracycline ...

Hyaluronidase and protease activities from Indian snake venoms: neutralization by Mimosa pudica root extract.

Science.gov (United States)

Girish, K S; Mohanakumari, H P; Nagaraju, S; Vishwanath, B S; Kemparaju, K

2004-06-01

The aqueous root extract of Mimosa pudica dose dependently inhibited the hyaluronidase and protease activities of Indian snakes (Naja naja, Vipera russelii and Echis carinatus) venom. Copyright 2004 Elsevier B.V.

Extracellular Neutrophil Proteases Are Efficient Regulators of IL-1, IL-33, and IL-36 Cytokine Activity but Poor Effectors of Microbial Killing.

Science.gov (United States)

Clancy, Danielle M; Sullivan, Graeme P; Moran, Hannah B T; Henry, Conor M; Reeves, Emer P; McElvaney, Noel G; Lavelle, Ed C; Martin, Seamus J

2018-03-13

Neutrophil granule proteases are thought to function as anti-microbial effectors, cooperatively hydrolyzing microorganisms within phagosomes, or upon deployment into the extracellular space. However, evidence also suggests that neutrophil proteases play an important role in the coordination and escalation of inflammatory reactions, but how this is achieved has been obscure. IL-1 family cytokines are important initiators of inflammation and are typically released via necrosis but require proteolytic processing for activation. Here, we show that proteases liberated from activated neutrophils can positively or negatively regulate the activity of six IL-1 family cytokines (IL-1α, IL-1β, IL-33, IL-36α, IL-36β, and IL-36γ) with exquisite sensitivity. In contrast, extracellular neutrophil proteases displayed very poor bactericidal activity, exhibiting 100-fold greater potency toward cytokine processing than bacterial killing. Thus, in addition to their classical role as phagocytes, neutrophils play an important immunoregulatory role through deployment of their granule proteases into the extracellular space to process multiple IL-1 family cytokines. Copyright © 2018 The Author(s). Published by Elsevier Inc. All rights reserved.

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

DEFF Research Database (Denmark)

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

2010-01-01

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

Reactivities of tetracycline and oxytetracycline with OH radicals

International Nuclear Information System (INIS)

Dziegielewski, J.O.; Glowacki, P.

1982-01-01

Decomposition yields of tetracycline sulphate and oxytetracycline sulphate were determined in argon saturated 0.1N H 2 SO 4 solutions. The decomposition yields of teracyclines decrease in the presence of the Cl - ions. The reaction rate constants of the OH radicals with tetracyclines were also determined. (author)

High throughput in vivo protease inhibitor selection platform

DEFF Research Database (Denmark)

2017-01-01

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

Regenerative potential and anti-bacterial activity of tetracycline loaded apatitic nanocarriers for the treatment of periodontitis

International Nuclear Information System (INIS)

Madhumathi, K; Sampath Kumar, T S

2014-01-01

Current treatment of periodontal infections includes mechanical debridement, administration of antibiotics and bone grafting. Oral administration of antibiotics results in undesirable side effects, while current modes of local administration are affected by problems concerning allergic response to the polymeric carrier agents. We have developed an osteoconductive drug delivery system composed of apatitic nanocarriers capable of providing sustained delivery of drugs in the periodontium. Calcium deficient hydroxyapatite (CDHA) nanocarriers of different Ca/P ratios were synthesized and characterized using the x-ray diffraction method, transmission electron microscopy, inductively coupled plasma atomic emission spectroscopy, Fourier transform infrared spectroscopy and the BET gas isotherm method. Loading and release studies performed with tetracycline showed a sustained release of up to 88% in phosphate buffered saline over a period of five days. Antibacterial activity studies showed that the tetracycline loaded CDHA (TC-CDHA) nanocarriers were effective against S. aureus and E. coli bacteria. The biocompatibility of the TC-CDHA nanocarriers was demonstrated using an alamar blue assay and further characterized by cell uptake studies. Interestingly, cell uptake of drug loaded CDHA also increased the cellular proliferation of human periodontal ligament fibroblast cells. Hence, it can be concluded that the CDHA nanocarriers are ideal drug delivery agents and have bone regenerative potential for local periodontal applications. (paper)

Two-Dimensional Zymography of Proteases from Steatotic Duck Liver.

Science.gov (United States)

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

2017-01-01

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

Heterologous expression of Hordeum vulgare cysteine protease in yeast

DEFF Research Database (Denmark)

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

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

Characterization of detergent compatible protease from halophilic Virgibacillus sp. CD6.

Science.gov (United States)

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

2018-02-01

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

Comparative Detection of Alkaline Protease Production in Exiguobacterium acetylicum

International Nuclear Information System (INIS)

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

2009-01-01

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

21 CFR 520.2345g - Tetracycline hydrochloride and sodium novobiocin tablets.

Science.gov (United States)

2010-04-01

... 21 Food and Drugs 6 2010-04-01 2010-04-01 false Tetracycline hydrochloride and sodium novobiocin... § 520.2345g Tetracycline hydrochloride and sodium novobiocin tablets. (a) Specifications. Each tablet contains the equivalent of 60 milligrams of tetracycline hydrochloride and 60 milligrams of novobiocin, or...

Reactivities of tetracycline and oxytetracycline with OH radicals

Energy Technology Data Exchange (ETDEWEB)

Dziegielewski, J O; Glowacki, P [Uniwersytet Slaski, Katowice (Poland)

1982-05-03

Decomposition yields of tetracycline sulphate and oxytetracycline sulphate were determined in argon saturated 0.1N H/sub 2/SO/sub 4/ solutions. The decomposition yields of teracyclines decrease in the presence of the Cl/sup -/ ions. The reaction rate constants of the OH radicals with tetracyclines were also determined.

Lyso-myristoyl phosphatidylcholine micelles sustain the activity of Dengue non-structural (NS) protein 3 protease domain fused with the full-length NS2B.

Science.gov (United States)

Huang, Qiwei; Li, Qingxin; Joy, Joma; Chen, Angela Shuyi; Ruiz-Carrillo, David; Hill, Jeffrey; Lescar, Julien; Kang, Congbao

2013-12-01

Dengue virus (DENV), a member of the flavivirus genus, affects 50-100 million people in tropical and sub-tropical regions. The DENV protease domain is located at the N-terminus of the NS3 protease and requires for its enzymatic activity a hydrophilic segment of the NS2B that acts as a cofactor. The protease is an important antiviral drug target because it plays a crucial role in virus replication by cleaving the genome-coded polypeptide into mature functional proteins. Currently, there are no drugs to inhibit DENV protease activity. Most structural and functional studies have been conducted using protein constructs containing the NS3 protease domain connected to a soluble segment of the NS2B membrane protein via a nine-residue linker. For in vitro structural and functional studies, it would be useful to produce a natural form of the DENV protease containing the NS3 protease domain and the full-length NS2B protein. Herein, we describe the expression and purification of a natural form of DENV protease (NS2BFL-NS3pro) containing the full-length NS2B protein and the protease domain of NS3 (NS3pro). The protease was expressed and purified in detergent micelles necessary for its folding. Our results show that this purified protein was active in detergent micelles such as lyso-myristoyl phosphatidylcholine (LMPC). These findings should facilitate further structural and functional studies of the protease and will facilitate drug discovery targeting DENV. Copyright © 2013 Elsevier Inc. All rights reserved.

Dimerization-Induced Allosteric Changes of the Oxyanion-Hole Loop Activate the Pseudorabies Virus Assemblin pUL26N, a Herpesvirus Serine Protease.

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Martin Zühlsdorf

2015-07-01

Full Text Available Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137; both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution.

Synergistic action of protease-modulating matrix and autologous growth factors in healing of diabetic foot ulcers. A prospective randomized trial.

Science.gov (United States)

Kakagia, Despoina D; Kazakos, Konstantinos J; Xarchas, Konstantinos C; Karanikas, Michael; Georgiadis, George S; Tripsiannis, Gregory; Manolas, Constantinos

2007-01-01

This study tests the hypothesis that addition of a protease-modulating matrix enhances the efficacy of autologous growth factors in diabetic ulcers. Fifty-one patients with chronic diabetic foot ulcers were managed as outpatients at the Democritus University Hospital of Alexandroupolis and followed up for 8 weeks. All target ulcers were > or = 2.5 cm in any one dimension and had been previously treated only with moist gauze. Patients were randomly allocated in three groups of 17 patients each: Group A was treated only with the oxidized regenerated cellulose/collagen biomaterial (Promogran, Johnson & Johnson, New Brunswick, NJ), Group B was treated only with autologous growth factors delivered by Gravitational Platelet Separation System (GPS, Biomet), and Group C was managed by a combination of both. All ulcers were digitally photographed at initiation of the study and then at change of dressings once weekly. Computerized planimetry (Texas Health Science Center ImageTool, Version 3.0) was used to assess ulcer dimensions that were analyzed for homogeneity and significance using the Statistical Package for Social Sciences, Version 13.0. Post hoc analysis revealed that there was significantly greater reduction of all three dimensions of the ulcers in Group C compared to Groups A and B (all P<.001). Although reduction of ulcer dimensions was greater in Group A than in Group B, these differences did not reach statistical significance. It is concluded that protease-modulating dressings act synergistically with autologous growth factors and enhance their efficacy in diabetic foot ulcers.

Enzymatic activity of proteases and its isoenzymes in fermentation process in cultivars of cocoa (Theobroma cacao L. produced in southern Bahia, Brazil

Directory of Open Access Journals (Sweden)

Luciane Santos SOUSA

Full Text Available Abstract The fermentation of cocoa seeds envolves microbial processes and the action of enzymes. To identify the possible differences in the cocoa fermentation process, with regards to proteolysis, this study has the objective of determining protease activity (under predetermined conditions and its isoenzymes in two cocoa cultivars (PH-16 and HRT-1188 in different cocoa fermentation times, in addition to establishing the microbial load (molds and yeasts and aerobic mesophilic. Protease and its isoenzymes were extracted and partially purified and the enzymatic activities determined by spectrophotometry. The results showed that the proteases activity was higher at 66h of fermentation for both cultivars. When the isoenzymes activity was evaluated, the results demonstrated similar activity behavior for both cultivars, with regards to the isoenzymes aminopeptidase and carboxypeptidase, although the behavior of the endoprotease isoenzyme activity proved to be a little different for TSH-1188 cultivar. Concerning microbiological analyses, the results indicate that the period after molds and yeast counting reduction is consistent with the period of protease activity increase.

Comparative activity of tigecycline and tetracycline on Gram-negative and Gram-positive bacteria revealed by a multicentre study in four North European countries

DEFF Research Database (Denmark)

Nilsson, Lennart E; Frimodt-Møller, Niels; Vaara, Martti

2011-01-01

This study involves a multicentre surveillance of tigecycline and tetracycline activity against Gram-negative and Gram-positive bacteria from primary care centres (PCCs), general hospital wards (GHWs) and intensive care units (ICUs) in Denmark (n = 9), Finland (n = 10), Norway (n = 7) and Sweden (n...

The antimicrobial and antifungal efficacy of tetracycline-integrated gutta-percha

Directory of Open Access Journals (Sweden)

Bodrumlu Emre

2008-01-01

Full Text Available The purpose of this study was in vitro evaluation of the antimicrobial and antifungal efficacy of commercially available gutta-percha containing tetracycline on some potential endodontic pathogens. The test microorganisms were Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus, Porphyromonas endodontalis, and Candida albicans . Tetracycline-integrated gutta-percha (TGP cones, tetracycline disc, and conventional gutta-percha cones of the same size were placed on the inoculated plates. The plates were incubated at 37°C aerobically or anaerobically. Growth inhibition zones on each plate were inspected at 24, 48, and 72 h. Tetracycline disc and TGP cones inhibited all the tested bacterial strains, however the greatest antimicrobial effect was seen on S. aureus. Tetracycline disc and TGP seemed less effective on E. faecalis and P. aeruginosa. However, all tested treatments were unable to affect C. albicans . Based on the results of this study, it seems that TGP offers an antimicrobial advantage over conventional gutta-percha.

Rapid method for measuring protease activity in milk using radiolabeled casein

International Nuclear Information System (INIS)

Christen, G.L.

1987-01-01

A rapid means to detect the presence of protease activity in raw milk could be useful in predicting keeping ability of products made from that milk. A 30-min assay has been developed and compared with three other methods of detecting protease. Casein, [methyl- 14 C]-methylated-alpha was purchased from a radioisotope supplier. Concentrations of substrate from 2 to 20 nCi gave counts per minute, which increased linearly when counted with the Charm analyzer. There was not a significant difference in counting times of 10, 20, or 30 min. A mixture of sodium acetate and acetic acid precipitated nonhydrolyzed substrate with an efficiency of 97%. Comparison of the [ 14 C] casein assay, a casein fluorescein isothiocyanate assay, trinitrobenzenesulfonic acid procedure, and the Hull procedure using protease from psychrotrophic bacteria revealed that the [ 14 C] casein and casein fluorescein isothiocyanate methods were roughly equivalent and that the radiometric procedure was 10 times more sensitive than the trinitrobenzenesulfonic acid assay. The radiometric procedure was approximately 10(4) times more sensitive than the Hull procedure. The [ 14 C] casein and casein fluorescein isothiocyanate methods were similar in time required, about 30 min, while the trinitrobenzenesulfonic acid assay and Hull method required about 1 h plus reagent preparation time. The [ 14 C] casein procedure was most expensive per test; the other three were cheaper and similar to each other in cost

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

Science.gov (United States)

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

2018-04-27

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

Oxidative Stress: Promoter of Allergic Sensitization to Protease Allergens?

NARCIS (Netherlands)

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

2017-01-01

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

Investigation of the Genetic Basis of Tetracycline Resistance in ...

African Journals Online (AJOL)

Purpose: To determine the prevalence and genetic basis of tetracycline resistance in Staphylococcus aureus. Methods: One hundred and thirty (130) clinical isolates of S. aureus were collected from Khyber Teaching. Hospital, Peshawar, Pakistan. Susceptibility to antibiotics (doxycycline, tetracycline and minocycline) was.

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

Directory of Open Access Journals (Sweden)

Jeong Hee Kim

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

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus.

Science.gov (United States)

Salles, Hévila Oliveira; Braga, Ana Carolina Linhares; Nascimento, Maria Thayana dos Santos Canuto do; Sousa, Ana Márjory Paiva; Lima, Adriano Rodrigues; Vieira, Luiz da Silva; Cavalcante, Antônio Cézar Rocha; Egito, Antonio Silvio do; Andrade, Lúcia Betânia da Silva

2014-01-01

Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (Albizia lebbeck, Ipomoea asarifolia, Jatropha curcas, Libidibia ferrea, Moringa oleifera and Ricinus communis (P0.05, Bonferroni test). Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

Lectin, hemolysin and protease inhibitors in seed fractions with ovicidal activity against Haemonchus contortus

Directory of Open Access Journals (Sweden)

Hévila Oliveira Salles

Full Text Available Bioactive molecules of plant species are promising alternatives for the chemical control of gastrointestinal nematodes in ruminants. Extracts of native and exotic seed species from Brazil's semi-arid region were tested in vitro in an egg hatch assay and the bioactivity of their proteins was investigated. Each seed species was subjected to three extractions with three types of solvents. All the seeds showed ovicidal activity, which varied according to the solvents. Higher ovicidal activity was found in the molecule fractions of low molecular weight (0.05, Bonferroni test. Hemagglutinating activity was detected in the fractions of C. spectabilis and M. oleifera fractions, hemolysin activity in the A. lebbeck and M. oleifera fractions, serine protease inhibitory activity in the A. lebbeck, I. asarifolia, J. curcas, M. oleifera and R. communis fractions, cysteine protease inhibitor activity in the M. oleifera fraction, and no protein activity in the L. ferrea fraction. The results of this work reveal new plant species with a potential for use in controlling nematode parasites in goats, thus opening a new field of research involving plant protein molecules with ovicidal properties.

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

Czech Academy of Sciences Publication Activity Database

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

2001-01-01

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

The Inflammatory Actions of Coagulant and Fibrinolytic Proteases in Disease

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Michael Schuliga

2015-01-01

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

Tetracycline removal during wastewater treatment in high-rate algal ponds

International Nuclear Information System (INIS)

Godos, Ignacio de; Muñoz, Raúl; Guieysse, Benoit

2012-01-01

Highlights: ► Tetracycline removal was most likely caused by photodegradation and biosorption. ► Tetracycline presence was linked to biomass deflocculation and poor settleability. ► Deflocculation did not impact treatment efficiency. ► Deflocculation may hamper biomass recover during full-scale treatment. - Abstract: With the hypothesis that light supply can impact the removal of veterinary antibiotics during livestock wastewater treatment in high rate algal ponds (HRAPs), this study was undertaken to determine the mechanisms of tetracycline removal in these systems. For this purpose, two HRAPs were fed with synthetic wastewater for 46 days before tetracycline was added at 2 mg L −1 to the influent of one of the reactors (Te-HRAP). From day 62, dissolved tetracycline removal stabilized around 69 ± 1% in the Te-HRAP and evidence from batch assays suggests that this removal was mainly caused by photodegradation and biosorption. Tetracycline addition was followed by the deflocculation of the Te-HRAP biomass but had otherwise no apparent impact on the removal of the chemical oxygen demand (COD) and biomass productivity. The results from the batch assays also suggested that the light-shading and/or pollutant-sequestrating effects of the biomass limited tetracycline removal in the pond. For the first time, these results demonstrate that the shallow geometry of HRAPs is advantageous to support the photodegradation of antibiotics during wastewater biological treatment but that the presence of these pollutants could hamper biomass recovery. These findings have significant implications for algal-based environmental biotechnologies and must be confirmed under field conditions.

Characterization of the protease activity that cleaves the extracellular domain of β-dystroglycan

International Nuclear Information System (INIS)

Zhong Di; Saito, Fumiaki; Saito, Yuko; Nakamura, Ayami; Shimizu, Teruo; Matsumura, Kiichiro

2006-01-01

Dystroglycan (DG) complex, composed of αDG and βDG, provides a link between the extracellular matrix (ECM) and cortical cytoskeleton. Although the proteolytic processing of βDG was reported in various physiological and pathological conditions, its exact mechanism remains unknown. In this study, we addressed this issue using the cell culture system of rat schwannoma cell line RT4. We found that the culture medium of RT4 cells was enriched with the protease activity that degrades the fusion protein construct of the extracellular domain of βDG specifically. This activity was suppressed by the inhibitor of matrix metalloproteinase-2 (MMP-2) and MMP-9, but not by the inhibitors of MMP-1, MMP-3, MMP-8, and MMP-13. Zymography and RT-PCR analysis showed that RT4 cells secreted MMP-2 and MMP-9 into the culture medium. Finally, active MMP-2 and MMP-9 enzymes degraded the fusion protein construct of the extracellular domain of βDG. These results indicate (1) that RT4 cells secrete the protease activity that degrades the extracellular domain of βDG specifically and (2) that MMP-2 and MMP-9 may be involved in this process

Sorption of tetracycline on organo-montmorillonites

International Nuclear Information System (INIS)

Liu, Niu; Wang, Ming-xia; Liu, Ming-ming; Liu, Fan; Weng, Liping; Koopal, Luuk K.; Tan, Wen-feng

2012-01-01

Highlights: ► The sorption capacity of tetracycline on Mont. modified with QACs was highly promoted. ► Tetracycline adsorbed on organoclay was affected by the amount and the length of QACs. ► Tetracycline adsorption on organoclay exhibited high pH-dependence below 5. - Abstract: Tetracycline (TC) is a veterinary antibiotic that is frequently detected as pollutant in the environment. Powerful adsorbents are required for removing TC. The present paper compares the TC adsorption capacity of Na-montmorillonite (Na-mont) with six organo-montmorillonites (organo-monts). Three quaternary ammonium cations (QACs) with different alkyl-chain lengths were used as modifiers. Powder X-ray diffraction indicated that the d 001 values of organo-monts increased with increasing the QACs loading and alkyl-chain length. The CECs of the organo-monts were substantially lower than that of Na-mont and decreased with QACs chain length and increased loading. The modeling of the adsorption kinetics revealed that the processes of TC adsorption on the tested samples could be well fitted by the pseudo-second-order equation. The maximum adsorption capacities of TC on the organo-monts (1000–2000 mmol/kg) were considerably higher than that on Na-mont (769 mmol/kg). Both the Langmuir and Freundlich model could fit the adsorption isotherms. The TC adsorption to the organo-monts increase significantly with decreasing the pH below 5.5 because of the electrostatic interaction, and a high QACs loading performed better than a low loading at around pH 3.

Purification and characterization of protease enzyme from ...

African Journals Online (AJOL)

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

Bacillus amyloliquefaciens SUBSP. plantarum PROBIOTIC STRAINS AS PROTEASE PRODUCERS

Directory of Open Access Journals (Sweden)

E. V. Маtseliukh

2015-04-01

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

Kinetics of tetracycline, oxytetracycline, and chlortetracycline adsorption and desorption on two acid soils

DEFF Research Database (Denmark)

Fernandez Calviño, David; Bermúdez-Couso, Alipio; Arias-Estévez, Manuel

2015-01-01

The purpose of this work was to quantify retention/release of tetracycline, oxytetracycline, and chlortetracycline on two soils, paying attention to sorption kinetics and to implications of the adsorption/desorption processes on transfer of these pollutants to the various environmental compartments...... tetracycline > oxytetracycline > chlortetracycline in soil 1, with similar values for the three antibiotics and the sequence tetracycline > chlortetracycline > oxytetracycline in soil 2. The desorption sequences were oxytetracycline > tetracycline > chlortetracycline in soil 1 and oxytetracycline...... > chlortetracycline > tetracycline in soil 2. In conclusion, the SFC technique has yielded new kinetic data regarding tetracycline, oxytetracycline, and chlortetracycline adsorption/desorption on soils, indicating that it can be used to shed further light on the retention and transport processes affecting antibiotics...

Effects of protease-activated receptor 1 inhibition on anxiety and fear following status epilepticus.

Science.gov (United States)

Bogovyk, Ruslan; Lunko, Oleksii; Fedoriuk, Mihail; Isaev, Dmytro; Krishtal, Oleg; Holmes, Gregory L; Isaeva, Elena

2017-02-01

Protease-activated receptor 1 (PAR1) is an important contributor to the pathogenesis of a variety of brain disorders associated with a risk of epilepsy development. Using the lithium-pilocarpine model of temporal lobe epilepsy (TLE), we recently showed that inhibition of this receptor during the first ten days after pilocarpine-induced status epilepticus (SE) results in substantial anti-epileptogenic and neuroprotective effects. As PAR1 is expressed in the central nervous system regions of importance for processing emotional reactions, including amygdala and hippocampus, and TLE is frequently associated with a chronic alteration of the functions of these regions, we tested the hypothesis that PAR1 inhibition could modulate emotionally driven behavioral responses of rats experiencing SE. We showed that SE induces a chronic decrease in the animals' anxiety-related behavior and an increase of locomotor activity. PAR1 inhibition after SE abolished the alteration of the anxiety level but does not affect the increase of locomotor activity in the open field and elevated plus maze tests. Moreover, while PAR1 inhibition produces an impairment of memory recall in the context fear conditioning paradigm in the control group, it substantially improves contextual and cued fear learning in rats experiencing SE. These data suggest that PAR1-dependent signaling is involved in the mechanisms underlying emotional disorders in epilepsy. Copyright © 2016 Elsevier Inc. All rights reserved.

Identification of SlpB, a Cytotoxic Protease from Serratia marcescens.

Science.gov (United States)

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

2015-07-01

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

Optimization of alkaline protease production and its fibrinolytic ...

African Journals Online (AJOL)

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

Isolation of Mucorales from processed maize (Zea mays L.) and screening for protease activity

OpenAIRE

de Azevedo Santiago, Andr? Luiz Cabral Monteiro; de Souza Motta, Cristina Maria

2008-01-01

Mucorales were isolated from maize flour, corn meal and cooked cornflakes using surface and depth plate methods. Rhizopus oryzae, Circinella muscae, Mucor subtilissimus, Mucor hiemalis f. hiemalis, Syncephalastrum racemosum, Rhizopus microsporus var. chinensis and Absidia cylindrospora showed protease activity.

Protease-activated receptor (PAR)-2 is required for PAR-1 signalling in pulmonary fibrosis

NARCIS (Netherlands)

Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

2015-01-01

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease of unknown aetiology. Compelling evidence suggests that both protease-activated receptor (PAR)-1 and PAR-2 participate in the development of pulmonary fibrosis. Previous studies have shown that bleomycin-induced

Pharmacological targeting of protease-activated receptor 2 affords protection from bleomycin-induced pulmonary fibrosis

NARCIS (Netherlands)

C. Lin (Cong); J. von der Thusen (Jan); J. Daalhuisen (Joost); M. Ten Brink (Marieke); B. Crestani (Bruno); T. van der Poll (Tom); K. Borensztajn (Keren); C. Arnold Spek (C.)

2015-01-01

textabstractIdiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed

Isolation of Mucorales from processed maize (Zea mays L.) and screening for protease activity

Science.gov (United States)

de Azevedo Santiago, André Luiz Cabral Monteiro; de Souza Motta, Cristina Maria

2008-01-01

Mucorales were isolated from maize flour, corn meal and cooked cornflakes using surface and depth plate methods. Rhizopus oryzae, Circinella muscae, Mucor subtilissimus, Mucor hiemalis f. hiemalis, Syncephalastrum racemosum, Rhizopus microsporus var. chinensis and Absidia cylindrospora showed protease activity. PMID:24031292

Partial characterization of cold active amylases and proteases of Streptomyces sp. from Antarctica

Directory of Open Access Journals (Sweden)

Mihaela Cotârleţ

2011-09-01

Full Text Available The aim of this study was to isolate novel enzyme-producing bacteria from vegetation samples from East Antarctica and also to characterize them genetically and biochemically in order to establish their phylogeny. The ability to grow at low temperature and to produce amylases and proteases cold-active was also tested. The results of the 16S rRNA gene sequence analysis showed that the 4 Alga rRNA was 100% identical to the sequences of Streptomyces sp. rRNA from Norway and from the Solomon Islands. The Streptomyces grew well in submerged system at 20ºC, cells multiplication up to stationary phase being drastically increased after 120 h of submerged cultivation. The beta-amylase production reached a maximum peak after seven days, while alpha-amylase and proteases were performing biosynthesis after nine days of submerged cultivation at 20ºC. Newly Streptomyces were able to produce amylase and proteases in a cold environment. The ability to adapt to low temperature of these enzymes could make them valuable ingredients for detergents, the food industry and bioremediation processes which require low temperatures.

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

Science.gov (United States)

Lai, Huiguo; Teramoto, Tadahisa; Padmanabhan, Radhakrishnan

2014-01-01

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

The protease inhibitor HAI-2, but not HAI-1, regulates matriptase activation and shedding through prostasin

DEFF Research Database (Denmark)

Friis, Stine; Sales, Katiuchia Uzzun; Schafer, Jeffrey Martin

2014-01-01

cells. Whereas ablation of HAI-1 did not affect matriptase in epithelial cells of the small or large intestine, ablation of HAI-2 resulted in the loss of matriptase from both tissues. Gene silencing studies in intestinal Caco-2 cell monolayers revealed that this loss of cell-associated matriptase......The membrane-anchored serine proteases, matriptase and prostasin, and the membrane-anchored serine protease inhibitors, hepatocyte growth factor activator inhibitor (HAI)-1 and HAI-2, are critical effectors of epithelial development and postnatal epithelial homeostasis. Matriptase and prostasin...... form a reciprocal zymogen activation complex that results in the formation of active matriptase and prostasin that are targets for inhibition by HAI-1 and HAI-2. Conflicting data, however, have accumulated as to the existence of auxiliary functions for both HAI-1 and HAI-2 in regulating...

Tunable protease-activatable virus nanonodes.

Science.gov (United States)

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

2014-05-27

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

Characterization and identification of proteases secreted by Aspergillus fumigatus using free flow electrophoresis and MS.

Science.gov (United States)

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

2009-06-01

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

Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

International Nuclear Information System (INIS)

Alvarez, Enrique; Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M.

2011-01-01

Highlights: → Novel role for poliovirus 2A protease as splicing modulator. → Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. → Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A pro modulating the alternative splicing of pre-mRNAs. Expression of 2A pro potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A pro abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A pro , leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A pro on splicing is to selectively block the second catalytic step.

Alternative splicing, a new target to block cellular gene expression by poliovirus 2A protease

Energy Technology Data Exchange (ETDEWEB)

Alvarez, Enrique, E-mail: ealvarez@cbm.uam.es [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain); Castello, Alfredo; Carrasco, Luis; Izquierdo, Jose M. [Centro de Biologia Molecular Severo Ochoa (CSIC-UAM), Nicolas Cabrera, 1 Universidad Autonoma de Madrid, Cantoblanco, 28049 Madrid (Spain)

2011-10-14

Highlights: {yields} Novel role for poliovirus 2A protease as splicing modulator. {yields} Poliovirus 2A protease inhibits the alternative splicing of pre-mRNAs. {yields} Poliovirus 2A protease blocks the second catalytic step of splicing. -- Abstract: Viruses have developed multiple strategies to interfere with the gene expression of host cells at different stages to ensure their own survival. Here we report a new role for poliovirus 2A{sup pro} modulating the alternative splicing of pre-mRNAs. Expression of 2A{sup pro} potently inhibits splicing of reporter genes in HeLa cells. Low amounts of 2A{sup pro} abrogate Fas exon 6 skipping, whereas higher levels of protease fully abolish Fas and FGFR2 splicing. In vitro splicing of MINX mRNA using nuclear extracts is also strongly inhibited by 2A{sup pro}, leading to accumulation of the first exon and the lariat product containing the unspliced second exon. These findings reveal that the mechanism of action of 2A{sup pro} on splicing is to selectively block the second catalytic step.

Purification and characterization of alkaline proteases from aspergillus terreus

International Nuclear Information System (INIS)

Hussain, A.; Mannan, A.; Zubair, H.; Mirza, B.

2010-01-01

Proteases belong to an important class of enzymes known as hydrolases and catalyze hydrolysis of proteins. They act primarily to degrade proteins that are used for energy production and as biosynthetic precursors. In the following study, protease produced from Aspergillus terreus was found to be thermo stable and included in the category of alkaline serine and metallo protease. During partial purification, presence of enzyme in 60% (NH/sub 4/)/sub 2/SO/sub 4/ indicated small molecular weight polypeptide; later purification with Sephadex G-75 fractionation yielded a single proteolytic active molecule. At final purification step, the increase in specific activity of the enzyme was 7.5 fold with 23% yield. SDS-PAGE analysis revealed that alkaline protease of Aspergillus terreus is a monomer with approximate molecular weight of 35 kDa. Optimum pH for protease activity was found in the range of 7.5-11.0 (maximum at pH 8.5), thus apparently classified as an alkaline protease. The enzyme was thermo stable towards high temperature (60 deg. C), however it denatured irreversibly at 70 deg. C showing 80% loss of activity. The maximum proteolytic activity was found at 40 deg. C. The enzyme was effectively inhibited by PMSF, EDTA and urea whereas iodoacetamide and thiourea did not result in any loss in activity while cysteine was found to be activator molecule. The study with metal ions Mg/sup +2/, Mn/sup +2/ and Fe/sup +3/ (1 mM each) showed minute stimulatory effects on enzyme activity. Co/sup +2/ and Ca/sup +2/ (1 mM) had neither excitatory nor inhibitory effect while Hg/sup +2/ and Cu/sup +2/ (1 mM) slightly reduced the enzyme activity. (author)

Autocatalytic activity and substrate specificity of the pestivirus N-terminal protease Npro

International Nuclear Information System (INIS)

Gottipati, Keerthi; Acholi, Sudheer; Ruggli, Nicolas; Choi, Kyung H.

2014-01-01

Pestivirus N pro is the first protein translated in the viral polypeptide, and cleaves itself off co-translationally generating the N-terminus of the core protein. Once released, N pro blocks the host's interferon response by inducing degradation of interferon regulatory factor-3. N pro' s intracellular autocatalytic activity and lack of trans-activity have hampered in vitro cleavage studies to establish its substrate specificity and the roles of individual residues. We constructed N pro -GFP fusion proteins that carry the authentic cleavage site and determined the autoproteolytic activities of N pro proteins containing substitutions at the predicted catalytic sites Glu22 and Cys69, at Arg100 that forms a salt bridge with Glu22, and at the cleavage site Cys168. Contrary to previous reports, we show that N pro' s catalytic activity does not involve Glu22, which may instead be involved in protein stability. Furthermore, N pro does not have specificity for Cys168 at the cleavage site even though this residue is conserved throughout the pestivirus genus. - Highlights: • N pro' s autoproteolysis is studied using N pro -GFP fusion proteins. • N-terminal 17 amino acids are dispensable without loss of protease activity. • The putative catalytic residue Glu22 is not involved in protease catalysis. • No specificity for Cys168 at the cleavage site despite evolutionary conservation. • N pro prefers small amino acids with non-branched beta carbons at the P1 position

Effect of Gastrointestinal Protease Digestion on Bioactivity of Marine Peptides

DEFF Research Database (Denmark)

Jensen, Ida-Johanne; Andersen, Lisa Lystbæk; Ossum, Carlo Gunnar

2014-01-01

executed without concerning subsequent digestion after intake and the aim of this work was hence to investigate how the in vitro antioxidative, antihypertensive and caspase activating activities of peptides are affected by digestion with gastrointestinal (GI) proteases. Five different fish protein...... hydrolysates were chosen to study the effect of in vitro digestion on bioactivity. The protein concentration decreased in all samples during digestion and the molecular weight distribution of the peptides shifted towards lower values. Thus, in vitro digestion with GI proteases resulted in a further degradation...... of the peptides obtained by hydrolysis. The antihypertensive effect increased in all samples after digestion with GI proteases whereas the antioxidative capacity decreased. The effect on the caspase activity depended on the proteases used in the preparation of hydrolysates. In conclusion, the caspase activity...

Biochemical characterization of a halophilic, alkalithermophilic protease from Alkalibacillus sp. NM-Da2.

Science.gov (United States)

Abdel-Hamed, Asmaa R; Abo-Elmatty, Dina M; Wiegel, Juergen; Mesbah, Noha M

2016-11-01

An extracellular, halophilic, alkalithermophilic serine protease from the halo-alkaliphilic Alkalibacillus sp. NM-Da2 was purified to homogeneity by ethanol precipitation and anion-exchange chromatography. The purified protease was a monomeric enzyme with an approximate molecular mass of 35 kDa and exhibited maximal activity at 2.7 M NaCl, pH 55 °C 9 and 56 °C. The protease showed great temperature stability, retaining greater than 80 % of initial activity after 2 h incubation at 55 °C. The protease was also extremely pH tolerant, retaining 80 % of initial activity at pH 55 °C 10.5 after 30 min incubation. Protease hydrolyzed complex substrates, displaying activity on yeast extract, tryptone, casein, gelatin and peptone. Protease activity was inhibited at casein concentrations greater than 1.2 mg/mL. The enzyme was stable and active in 40 % (v/v) solutions of isopropanol, ethanol and benzene and was stable in the presence of the polysorbate surfactant Tween 80. Activity was stimulated with the oxidizing agent hydrogen peroxide. Inhibition with phenyl methylsulfonylfluoride indicates it is a serine protease. Synthetic saline wastewater treated with the protease showed 50 % protein removal after 5 h. Being halophilic, alkaliphilic and thermophilic, in addition to being resistant to organic solvents, this protease has potential for various applications in biotechnological and pharmaceutical industries.

Pharmacological Targeting of Protease-Activated Receptor 2 Affords Protection from Bleomycin-Induced Pulmonary Fibrosis

NARCIS (Netherlands)

Lin, Cong; von der Thüsen, Jan; Daalhuisen, Joost; ten Brink, Marieke; Crestani, Bruno; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

2015-01-01

Idiopathic pulmonary fibrosis is the most devastating diffuse fibrosing lung disease that remains refractory to therapy. Despite increasing evidence that protease-activated receptor 2 (PAR-2) contributes to fibrosis, its importance in pulmonary fibrosis is under debate. We addressed whether PAR-2

Organ distribution of sup(99m)Tc-Sn tetracycline antibiotics in rats

International Nuclear Information System (INIS)

Kalincak, M.; Machan, V.; Barna, K.

1976-01-01

The organ distribution of [sup(99m)Tc-Sn]tetracycline hydrochloride, [sup(99m)Tc-Sn]oxytetracycline hydrochloride and [sup(99m)Tc-Sn]rolitetracycline nitrate was studied in rats. It was shown that these preparations have a very similar organ distribution and are predominantly deposited in the kidneys. The maximum renal radioactivity level was found 2 to 4 hours after intravenous administration of the preparation, this, for [sup(99m)Tc-Sn]tetracycline hydrochloride 18.60%; for [sup(99m)Tc-Sn]oxytetracycline hydrochloride 17.09.=.; for [sup(99m)Tc-Sn]rolitetracycline nitrate 20.12%. The activity levels in the muscle, liver, heart, brain, lungs, stomach and spleen are minimal. (author)

Organ distribution of /sup 99m/Tc--Sn tetracycline antibiotics in rats

Energy Technology Data Exchange (ETDEWEB)

Kalincak, M; Machan, V; Barna, K [Univerzita P.J. Safarika, Kosice (Czechoslovakia). Lekarska Fakulta

1976-06-01

The organ distribution of (sup(99m)Tc-Sn)tetracycline hydrochloride, (sup(99m)Tc-Sn)oxytetracycline hydrochloride and (sup(99m)Tc-Sn)rolitetracycline nitrate was studied in rats. It was shown that these preparations have a very similar organ distribution and are predominantly deposited in the kidneys. The maximum renal radioactivity level was found 2 to 4 hours after intravenous administration of the preparation, this, for (sup(99m)Tc-Sn)tetracycline hydrochloride 18.60%; for (sup(99m)Tc-Sn)oxytetracycline hydrochloride 17.09.=.; for (sup(99m)Tc-Sn)rolitetracycline nitrate 20.12%. The activity levels in the muscle, liver, heart, brain, lungs, stomach and spleen are minimal.

Characterization of Fibrinolytic Proteases from Gloydius blomhoffii siniticus Venom

Directory of Open Access Journals (Sweden)

Suk Ho Choi

2011-09-01

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

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

International Nuclear Information System (INIS)

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

2011-01-01

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

Antioxidant and ACE-inhibitory activities of hemp (Cannabis sativa L.) protein hydrolysates produced by the proteases AFP, HT, Pro-G, actinidin and zingibain.

Science.gov (United States)

Teh, Sue-Siang; Bekhit, Alaa El-Din A; Carne, Alan; Birch, John

2016-07-15

Hemp protein isolates (HPIs) were hydrolysed by proteases (AFP, HT, ProG, actinidin and zingibain). The enzymatic hydrolysis of HPIs was evaluated through the degree of hydrolysis and SDS-PAGE profiles. The bioactive properties of the resultant hydrolysates (HPHs) were accessed through ORAC, DPPḢ scavenging and ACE-inhibitory activities. The physical properties of the resultant HPHs were evaluated for their particle sizes, zeta potential and surface hydrophobicity. HT had the highest rate of caseinolytic activity at the lowest concentration (0.1 mg mL(-1)) compared to other proteases that required concentration of 100 mg mL(-1) to achieve their maximum rate of caseinolytic activity. This led to the highest degree of hydrolysis of HPIs by HT in the SDS-PAGE profiles. Among all proteases and substrates, HT resulted in the highest bioactivities (ORAC, DPPḢ scavenging and ACE-inhibitory activities) generated from alkali extracted HPI in the shortest time (2 h) compared to the other protease preparations. Copyright © 2016 Elsevier Ltd. All rights reserved.

Conditional and inducible transgene expression in endothelial and hematopoietic cells using Cre/loxP and tetracycline-off systems.

Science.gov (United States)

Liu, Ju; Deutsch, Urban; Fung, Iris; Lobe, Corrinne G

2014-11-01

In the present study, the tetracycline-off and Cre/ loxP systems were combined to gain temporal and spatial control of transgene expression. Mice were generated that carried three transgenes: Tie2-tTA, tet-O-Cre and either the ZEG or ZAP reporter. Tie2-tTA directs expression of tetracycline-controlled transactivator (tTA) in endothelial and hematopoietic cells under the control of the Tie2 promoter. Tet-O-Cre produces Cre recombinase from a minimal promoter containing the tet-operator (tetO). ZEG or ZAP contains a strong promoter and a loxP -flanked stop sequence, followed by an enhanced green fluorescence protein (EGFP) or human placental alkaline phosphatase (hPLAP) reporter. In the presence of tetracycline, the tTA transactivator produced by Tie-2-tTA is disabled and Cre is not expressed. In the absence of tetracycline, the tTA binds tet-O-Cre to drive the expression of Cre, which recombines the loxP sites of the ZEG or ZAP transgene and results in reporter gene expression. In the present study, the expression of the ZEG or ZAP reporter genes in embryos and adult animals with and without tetracycline treatment was examined. In the presence of tetracycline, no reporter gene expression was observed. When tetracycline was withdrawn, Cre excision was activated and the reporter genes were detected in endothelial and hematopoietic cells. These results demonstrate that this system may be used to bypass embryonic lethality and access adult phenotypes.

Chemically modified tetracyclines stimulate matrix metalloproteinase-s production by periodontal ligament cells

NARCIS (Netherlands)

Bildt, M.M.; Snoek-van Beurden, A.M.P.; Groot, J. de; El, B. van; Kuijpers-Jagtman, A.M.; Hoff, J.W. van den

2006-01-01

Background and Objective: The purpose of this study was to investigate the effects of chemically modified tetracyclines (CMTs) on the production of gelatinases [matrix metalloproteinase (MMP)-2 and -9] by human periodontal ligament (PDL) cells, and on the activity of recombinant gelatinases.

Comparison of the efficacy of ethylenediaminetetraacetic acid and tetracycline hydrochloride as root conditioning agents: An in vitro study

Directory of Open Access Journals (Sweden)

Tarun Nanda

2012-01-01

Full Text Available Objective: Root surfaces of periodontitis-affected teeth are hypermineralized and contaminated with cytotoxic and other biologically active substances. Various root conditioning agents have been recommended as an adjunct to mechanical root surface debridement to expose dentin collagen and cementum bound proteins. The aim of the present in vitro study was to compare the efficacy of ethylenediaminetetraacetic acid (EDTA and tetracycline hydrochloride (HCl as root conditioning agents on planed root surfaces. Materials and Methods: A total of 20 human maxillary anterior teeth indicated for extraction due to chronic periodontitis were collected and root planed. The teeth were sectioned and specimens were divided into two groups - Group I and II. Group I dentin specimens were treated with EDTA and Group II specimens were treated with tetracycline HCl solution at a concentration of 10% by active burnishing technique for 3 min. The root surface samples were then examined by scanning electron microscope. Results: The results of this study showed that EDTA and tetracycline HCl were equally effective in removing the smear layer. It was observed that the total and patent dentinal tubules were more in number in teeth treated with tetracycline when compared to EDTA group. However, EDTA was found to be much more effective as root conditioning agent because it enlarged the diameter of dentinal tubules more than that of tetracycline HCl. Conclusion: The results of in vitro study showed that both the agents are good root conditioning agents if applied in addition to periodontal therapy. However, further studies are required to establish the in vivo importance of EDTA and tetracycline HCL as root conditioners.

Rapid colorimetric sensing of tetracycline antibiotics with in situ growth of gold nanoparticles

International Nuclear Information System (INIS)

Shen, Li; Chen, Jing; Li, Na; He, Pingli; Li, Zhen

2014-01-01

Highlights: • Tetracyclines directly reduce aurate into gold nanoparticles. • Gold nanoparticles showed characteristic plamson absorbance at 526 nm. • Quantitative detection of tetracyclines with the colorimetric assay. • Tetracyclines spiked urine samples can be detected with the assay. - Abstract: A colorimetric assay utilizing the formation of gold nanoparticles was developed to detect tetracycline antibiotics in fluidic samples. Tetracycline antibiotics showed the capability of directly reducing aurate salts into atomic gold which form gold nanoparticles spontaneously under proper conditions. The resulted gold nanoparticles showed characteristic plasmon absorbance at 526 nm, which can be visualized by naked eyes or with a spectrophotometer. UV–vis absorbance of the resulted gold nanoparticles is correlated directly with the concentrations of tetracycline antibiotics in the solution, allowing for quantitative colorimetric detection of tetracycline antibiotics. Reaction conditions, such as pH, temperature, reaction time, and ionic strength were optimized. Sensitivity of the colorimetric assay can be enhanced by the addition of gold nanoparticle seeds, a LOD as low as 20 ng mL −1 can be achieved with the help of seed particles. The colorimetric assay showed minimum interference from ethanol, methanol, urea, glucose, and other antibiotics such as sulfonamides, amino glycosides etc. Validity of the method was also evaluated on urine samples spiked with tetracycline antibiotics. The method provides a broad spectrum detection method for rapid and sensitive detection of reductive substances such as tetracycline antibiotics in liquid and biological samples

Rapid colorimetric sensing of tetracycline antibiotics with in situ growth of gold nanoparticles

Energy Technology Data Exchange (ETDEWEB)

Shen, Li [Logistics School, Beijing Wuzi University, Beijing 101149 (China); Chen, Jing; Li, Na [Logistics School, Beijing Wuzi University, Beijing 101149 (China); He, Pingli [State Key Laboratory of Animal Nutrition, China Agricultural University, Beijing 100094 (China); Li, Zhen [State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100193 (China)

2014-08-11

Highlights: • Tetracyclines directly reduce aurate into gold nanoparticles. • Gold nanoparticles showed characteristic plamson absorbance at 526 nm. • Quantitative detection of tetracyclines with the colorimetric assay. • Tetracyclines spiked urine samples can be detected with the assay. - Abstract: A colorimetric assay utilizing the formation of gold nanoparticles was developed to detect tetracycline antibiotics in fluidic samples. Tetracycline antibiotics showed the capability of directly reducing aurate salts into atomic gold which form gold nanoparticles spontaneously under proper conditions. The resulted gold nanoparticles showed characteristic plasmon absorbance at 526 nm, which can be visualized by naked eyes or with a spectrophotometer. UV–vis absorbance of the resulted gold nanoparticles is correlated directly with the concentrations of tetracycline antibiotics in the solution, allowing for quantitative colorimetric detection of tetracycline antibiotics. Reaction conditions, such as pH, temperature, reaction time, and ionic strength were optimized. Sensitivity of the colorimetric assay can be enhanced by the addition of gold nanoparticle seeds, a LOD as low as 20 ng mL{sup −1} can be achieved with the help of seed particles. The colorimetric assay showed minimum interference from ethanol, methanol, urea, glucose, and other antibiotics such as sulfonamides, amino glycosides etc. Validity of the method was also evaluated on urine samples spiked with tetracycline antibiotics. The method provides a broad spectrum detection method for rapid and sensitive detection of reductive substances such as tetracycline antibiotics in liquid and biological samples.

Optimization of alkaline protease production from Pseudomonas ...

African Journals Online (AJOL)

PRECIOUS

2009-12-15

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

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

Science.gov (United States)

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

2013-01-01

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

Effects of temperature, pH and NaCl on protease activity in digestive tract of young turbot, Scophthalmus maximus

Science.gov (United States)

Chen, Muyan; Zhang, Xiumei; Gao, Tianxiang; Chen, Chao

2006-09-01

The protease activity in digestive tract of young turbot Scophthalmus maximum was studied, and the optimal pH, temperature and NaCl concentration were determined for different portions of the fish's internal organs. The optimal activity in the fish's stomach was at pH of 2.2, while that in the intestinal extracts was within the alkaline range from 9.5 to 10.0. In hepatopancreas, the optimal pH was in low alkalinity at 8.5. The optimal reaction temperature was above 40°C in stomach, intestine and hepatopancreas. With increasing temperature, the pH value increased in stomach, while in the intestine, an opposite tendency was observed due to combined effect of pH and temperature. NaCl concentration showed inhibitory impact on protein digestion in hepatopancreas. The main protease for protein digestion in turbot seemed to be pepsin. Moreover, the maximum protease activity in different segments of intestine existed in the hindgut.

Production of alkaline proteases by alkalophilic Bacillus subtilis ...

African Journals Online (AJOL)

Tuoyo Aghomotsegin

2016-11-23

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

Performance and microbial community variations of anaerobic digesters under increasing tetracycline concentrations

KAUST Repository

Xiong, Yanghui; Harb, Moustapha; Hong, Pei-Ying

2017-01-01

The impact of different concentrations of tetracycline on the performance of anaerobic treatment was evaluated. Results revealed that for all of the tested tetracycline concentrations, no major sustained impact on methane production was observed. Instead, a significant increase in propionic acid was observed in the reactor subjected to the highest concentration of tetracycline (20 mg/L). Microbial community analyses suggest that an alternative methanogenic pathway, specifically that of methanol-utilizing methanogens, may be important for ensuring the stability of methane production in the presence of high tetracycline concentrations. In addition, the accumulation of propionate was due to an increase in volatile fatty acids (VFA)-producing bacteria coupled with a reduction in propionate utilizers. An increase in the abundance of tetracycline resistance genes associated with ribosomal protection proteins was observed after 30 days of exposure to high concentrations of tetracycline, while other targeted resistance genes showed no significant changes. These findings suggest that anaerobic treatment processes can robustly treat wastewater with varying concentrations of antibiotics while also deriving value-added products and minimizing the dissemination of associated antibiotic resistance genes.

Performance and microbial community variations of anaerobic digesters under increasing tetracycline concentrations

KAUST Repository

Xiong, Yanghui

2017-04-01

The impact of different concentrations of tetracycline on the performance of anaerobic treatment was evaluated. Results revealed that for all of the tested tetracycline concentrations, no major sustained impact on methane production was observed. Instead, a significant increase in propionic acid was observed in the reactor subjected to the highest concentration of tetracycline (20 mg/L). Microbial community analyses suggest that an alternative methanogenic pathway, specifically that of methanol-utilizing methanogens, may be important for ensuring the stability of methane production in the presence of high tetracycline concentrations. In addition, the accumulation of propionate was due to an increase in volatile fatty acids (VFA)-producing bacteria coupled with a reduction in propionate utilizers. An increase in the abundance of tetracycline resistance genes associated with ribosomal protection proteins was observed after 30 days of exposure to high concentrations of tetracycline, while other targeted resistance genes showed no significant changes. These findings suggest that anaerobic treatment processes can robustly treat wastewater with varying concentrations of antibiotics while also deriving value-added products and minimizing the dissemination of associated antibiotic resistance genes.

Performance and microbial community variations of anaerobic digesters under increasing tetracycline concentrations.

Science.gov (United States)

Xiong, Yanghui; Harb, Moustapha; Hong, Pei-Ying

2017-07-01

The impact of different concentrations of tetracycline on the performance of anaerobic treatment was evaluated. Results revealed that for all of the tested tetracycline concentrations, no major sustained impact on methane production was observed. Instead, a significant increase in propionic acid was observed in the reactor subjected to the highest concentration of tetracycline (20 mg/L). Microbial community analyses suggest that an alternative methanogenic pathway, specifically that of methanol-utilizing methanogens, may be important for ensuring the stability of methane production in the presence of high tetracycline concentrations. In addition, the accumulation of propionate was due to an increase in volatile fatty acids (VFA)-producing bacteria coupled with a reduction in propionate utilizers. An increase in the abundance of tetracycline resistance genes associated with ribosomal protection proteins was observed after 30 days of exposure to high concentrations of tetracycline, while other targeted resistance genes showed no significant changes. These findings suggest that anaerobic treatment processes can robustly treat wastewater with varying concentrations of antibiotics while also deriving value-added products and minimizing the dissemination of associated antibiotic resistance genes.

Cysteine Protease Zymography: Brief Review.

Science.gov (United States)

Wilkesman, Jeff

2017-01-01

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

Lon protease affects the RdxA nitroreductase activity and metronidazole susceptibility in Helicobacter pylori.

Science.gov (United States)

Tu, I-Fan; Liao, Jiahn-Haur; Yang, Feng-Ling; Lin, Nien-Tsung; Chan, Hong-Lin; Wu, Shih-Hsiung

2014-10-01

The lon gene of Helicobacter pylori strains is constitutively expressed during growth. However, virtually nothing is understood concerning the role of Lon in H. pylori. This study examined the function and physiological role of Lon in H. pylori (HpLon) using a trapping approach to identify putative Lon binding partners in the bacterium. Protease-deficient Lon was expressed and served as the bait in trapping approach to capture the interacting partners in H. pylori. The antibiotic susceptibility of wild-type and lon derivative mutants was determined by the E test trips and the disc diffusion assay. The effect of HpLon on RdxA activity was detected the change in NADPH oxidation and metronidazole reduction by spectrophotometer. Lon in Helicobacter pylori (HpLon) interacting partners are mostly associated with metronidazole activation. lon mutant presents more susceptible to metronidazole than that of the wild type, and this phenotype is recovered by complementation of the wild-type Lon. We found that the ATPases associated with a variety of cellular activities (AAA(+) ) module of HpLon causes a decrease in both NADPH oxidase and Mtz reductase activity in RdxA, a major Mtz-activating enzyme in H. pylori. Metronidazole resistance of H. pylori causes the serious medical problem worldwide. In this study, HpLon is involved in metronidazole susceptibility among H. pylori strains. We provide the evidence that HpLon alters RdxA activity in vitro. The decrease in metronidazole activation caused by HpLon is possibly prior to accumulate mutation in rdxA gene before the metronidazole-resistant strains to be occurred. © 2014 John Wiley & Sons Ltd.

Post-secretional activation of Protease IV by quorum sensing in Pseudomonas aeruginosa

OpenAIRE

Oh, Jungmin; Li, Xi-Hui; Kim, Soo-Kyong; Lee, Joon-Hee

2017-01-01

Protease IV (PIV), a key virulence factor of Pseudomonas aeruginosa is a secreted lysyl-endopeptidase whose expression is induced by quorum sensing (QS). We found that PIV expressed in QS mutant has severe reduction of activity in culture supernatant (CS), even though it is overexpressed to high level. PIV purified from the QS mutant (M-PIV) had much lower activity than the PIV purified from wild type (P-PIV). We found that the propeptide cleaved from prepro-PIV was co-purified with M-PIV, bu...

Purification and characterization of protease enzyme from ...

African Journals Online (AJOL)

user

2013-03-20

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

Relation between tetR and tetA expression in tetracycline resistant Escherichia coli

DEFF Research Database (Denmark)

Møller, Thea S. B.; Overgaard, Martin; Nielsen, Søren S.

2016-01-01

Background: Tetracyclines are among the most used antibiotics in livestock worldwide. Resistance is widely disseminated in Escherichia coli, where it is generally mediated by tetracycline efflux pumps, such as TetA. Expression of tetracycline efflux pumps is tightly controlled by the repressor Tet...

Differential Response of Extracellular Proteases of Trichoderma Harzianum Against Fungal Phytopathogens.

Science.gov (United States)

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

2016-09-01

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

Sonocatalytic Degradation of Antibiotics Tetracycline by Mn-Modified Diatomite

OpenAIRE

Guo, Yiping; Mi, Xiao; Li, Guoting; Chen, Xi

2017-01-01

Mn-modified diatomite was prepared by wet impregnation and subsequent calcinations processes. It was used as catalyst for sonocatalytic degradation of antibiotics tetracycline. Characterizations by scanning electron microscopy and X-ray diffraction pattern showed that the morphology and crystal structure of the modified diatomite were similar to these of raw diatomite. Despite containing very limited amount of Mn oxides, the Mn-modified diatomite showed much higher sonocatalytic activity than...

Hyper production of alkaline protease by mutagenized bacillus subtilis

International Nuclear Information System (INIS)

Qureshi, A.M.; Tanseem, F.

2010-01-01

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

Targeting protease activated receptor-1 with P1pal-12 limits bleomycin-induced pulmonary fibrosis

NARCIS (Netherlands)

Lin, Cong; Duitman, Janwillem; Daalhuisen, Joost; ten Brink, Marieke; von der Thüsen, Jan; van der Poll, Tom; Borensztajn, Keren; Spek, C. Arnold

2014-01-01

Idiopathic pulmonary fibrosis is the most devastating fibrotic diffuse parenchymal lung disease which remains refractory to pharmacological therapies. Therefore, novel treatments are urgently required. Protease-activated receptor (PAR)-1 is a G-protein-coupled receptor that mediates critical

HPLC method validation for modernization of the tetracycline hydrochloride capsule USP monograph

Directory of Open Access Journals (Sweden)

Emad M. Hussien

2014-12-01

Full Text Available This paper is a continuation to our previous work aiming at development and validation of a reversed-phase HPLC for modernization of tetracycline-related USP monographs and the USP general chapter . Previous results showed that the method is accurate and precise for the assay of tetracycline hydrochloride and the limit of 4-epianhydrotetracycline impurity in the drug substance and oral suspension monographs. The aim of the current paper is to examine the feasibility of the method for modernization of USP tetracycline hydrochloride capsule monograph. Specificity, linearity, accuracy and precision were examined for tetracycline hydrochloride assay and 4-epianhydrotetracycline limit. The method was linear in the concentration range from 80% to 160% (r>0.9998 of the assay concentration (0.1 mg/mL for tetracycline hydrochloride and from 50% to 150% (r>0.997 of the acceptance criteria specified in tetracycline hydrochloride capsule monograph for 4-epianhydrotetracycline (NMT 3.0%. The recovery at three concentration levels for tetracycline hydrochloride assay was between 99% and 101% and the RSD from six preparations at the concentration 0.1 mg/mL is less than 0.6%. The recovery for 4-epianhydrotetracycline limit procedure over the concentration range from 50% to 150% is between 96% and 102% with RSD less than 5%. The results met the specified acceptance criteria.

Thermolysin damages animal life through degradation of plasma proteins enhanced by rapid cleavage of serpins and activation of proteases.

Science.gov (United States)

Kong, Lulu; Lu, Anrui; Guan, Jingmin; Yang, Bing; Li, Muwang; Hillyer, Julián F; Ramarao, Nalini; Söderhäll, Kenneth; Liu, Chaoliang; Ling, Erjun

2015-01-01

Thermolysin, a metallopeptidase secreted by pathogenic microbes, is concluded as an important virulence factor due to cleaving purified host proteins in vitro. Using the silkworm Bombyx mori as a model system, we found that thermolysin injection into larvae induces the destruction of the coagulation response and the activation of hemolymph melanization, which results in larval death. Thermolysin triggers the rapid degradation of insect and mammalian plasma proteins at a level that is considerably greater than expected in vitro and/or in vivo. To more specifically explore the mechanism, thermolysin-induced changes to key proteins belonging to the insect melanization pathway were assessed as a window for observing plasma protein cleavage. The application of thermolysin induced the rapid cleavage of the melanization negative regulator serpin-3, but did not directly activate the melanization rate-limiting enzyme prophenoloxidase (PPO) or the terminal serine proteases responsible for PPO activation. Terminal serine proteases of melanization are activated indirectly after thermolysin exposure. We hypothesize that thermolysin induces the rapid degradation of serpins and the activation of proteases directly or indirectly, boosting uncontrolled plasma protein degradation in insects and mammalians. © 2014 Wiley Periodicals, Inc.

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

Science.gov (United States)

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

2013-11-01

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

HIV protease drug resistance and its impact on inhibitor design.

Science.gov (United States)

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

1999-07-01

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

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

Directory of Open Access Journals (Sweden)

Marian Dorcas Quain

2013-08-01

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

Regulator of G protein signaling 2 (RGS2 and RGS4 form distinct G protein-dependent complexes with protease activated-receptor 1 (PAR1 in live cells.

Directory of Open Access Journals (Sweden)

Sungho Ghil

Full Text Available Protease-activated receptor 1 (PAR1 is a G-protein coupled receptor (GPCR that is activated by natural proteases to regulate many physiological actions. We previously reported that PAR1 couples to Gi, Gq and G12 to activate linked signaling pathways. Regulators of G protein signaling (RGS proteins serve as GTPase activating proteins to inhibit GPCR/G protein signaling. Some RGS proteins interact directly with certain GPCRs to modulate their signals, though cellular mechanisms dictating selective RGS/GPCR coupling are poorly understood. Here, using bioluminescence resonance energy transfer (BRET, we tested whether RGS2 and RGS4 bind to PAR1 in live COS-7 cells to regulate PAR1/Gα-mediated signaling. We report that PAR1 selectively interacts with either RGS2 or RGS4 in a G protein-dependent manner. Very little BRET activity is observed between PAR1-Venus (PAR1-Ven and either RGS2-Luciferase (RGS2-Luc or RGS4-Luc in the absence of Gα. However, in the presence of specific Gα subunits, BRET activity was markedly enhanced between PAR1-RGS2 by Gαq/11, and PAR1-RGS4 by Gαo, but not by other Gα subunits. Gαq/11-YFP/RGS2-Luc BRET activity is promoted by PAR1 and is markedly enhanced by agonist (TFLLR stimulation. However, PAR1-Ven/RGS-Luc BRET activity was blocked by a PAR1 mutant (R205A that eliminates PAR1-Gq/11 coupling. The purified intracellular third loop of PAR1 binds directly to purified His-RGS2 or His-RGS4. In cells, RGS2 and RGS4 inhibited PAR1/Gα-mediated calcium and MAPK/ERK signaling, respectively, but not RhoA signaling. Our findings indicate that RGS2 and RGS4 interact directly with PAR1 in Gα-dependent manner to modulate PAR1/Gα-mediated signaling, and highlight a cellular mechanism for selective GPCR/G protein/RGS coupling.

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

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Madhusudhan Budatha

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

Functional protease profiling for diagnosis of malignant disease.

Science.gov (United States)

Findeisen, Peter; Neumaier, Michael

2012-01-01

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

Biotransformation of tetracycline by a novel bacterial strain Stenotrophomonas maltophilia DT1.

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Leng, Yifei; Bao, Jianguo; Chang, Gaofeng; Zheng, Han; Li, Xingxing; Du, Jiangkun; Snow, Daniel; Li, Xu

2016-11-15

Although several abiotic processes have been reported that can transform antibiotics, little is known about whether and how microbiological processes may degrade antibiotics in the environment. This work isolated one tetracycline degrading bacterial strain, Stenotrophomonas maltophilia strain DT1, and characterized the biotransformation of tetracycline by DT1 under various environmental conditions. The biotransformation rate was the highest when the initial pH was 9 and the reaction temperature was at 30°C, and can be described using the Michaelis-Menten model under different initial tetracycline concentrations. When additional substrate was present, the substrate that caused increased biomass resulted in a decreased biotransformation rate of tetracycline. According to disk diffusion tests, the biotransformation products of tetracycline had lower antibiotic potency than the parent compound. Six possible biotransformation products were identified, and a potential biotransformation pathway was proposed that included sequential removal of N-methyl, carbonyl, and amine function groups. Results from this study can lead to better estimation of the fate and transport of antibiotics in the environment and has the potential to be utilized in designing engineering processes to remove tetracycline from water and soil. Copyright © 2016 Elsevier B.V. All rights reserved.

Extracellular proteases of Trichoderma species. A review.

Science.gov (United States)

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

2005-01-01

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

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

International Nuclear Information System (INIS)

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

2016-01-01

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

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

Energy Technology Data Exchange (ETDEWEB)

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

2016-09-15

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

Expression and Characterization of Coprothermobacter proteolyticus Alkaline Serine Protease

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Tanveer Majeed

2013-01-01

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

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

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Zongyun Chen

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

Studies on the Catalytic Properties of Partially Purified Alkaline Proteases from Some Selected Microorganisms

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Titilayo Olufunke Femi-Ola

2012-09-01

Full Text Available Aims: The research was done to study the conditions enhancing catalytic activities of alkaline proteases from Vibro sp., Lactobacillus brevis, Zymomonas sp., Athrobacter sp., Corynebacterium sp. and Bacillus subtilis.Methodology and Results: The proteolytic enzymes were purified in 2-step procedures involving ammonium sulphate precipitation and sephadex G-150 gel permeation chromatography. The upper and lower limits for the specific activities of proteases from the selected microorganisms were estimated at 20.63 and 47.51 units/mg protein with Zymomonas protease having the highest specific activity towards casein as its substrate and purification fold of 3.46, while that ofLactobacillus brevis protease was 8.06. The native molecular weights of these active proteins ranged from 30.4 to 45.7 kDa with Athrobacter sp. protease having the highest weight for its subunits. The proteolytic enzymes had optimum pH range of 8 to 10 and temperature range of 50 to 62 ºC accounting for the percentage relative activity range of 75 to 94% and 71 to 84 % respectively. The activities of Lactobacillus brevis and Bacillus subtilis proteases were maximum at pH 9 and 10 respectively. Lactobacillus brevis protease activity was maximum at temperature of 62 ºC, while beyond this value, a general thermal instability of these active proteins was observed. At above 70 ºC, the catalytic activities of Corynebacterium sp., Vibrio sp., Zymomonas sp. and Arthrobacter sp. proteases were progressively reduced over a period of 120 min of incubation, while Bacillus subtlis and Lactobacillus brevis proteases were relatively stable. Effect of metal ions was investigated on the catalytic activity of protease from the microorganisms. Lactobacillus brevis,Zymomonas sp., Arthrobacter sp., Corynebacterium sp. and Bacillus subtilis protease activities were strongly activated by metal ions such as Ca+2 and Mg+2. Enzyme activities were inhibited strongly by Cu2+ and Hg2+ but were not

Proteases and antiproteases in chronic neutrophilic lung disease - relevance to drug discovery.

LENUS (Irish Health Repository)

Greene, Catherine M

2009-10-01

Chronic inflammatory lung diseases such as cystic fibrosis and emphysema are characterized by higher-than-normal levels of pulmonary proteases. While these enzymes play important roles such as bacterial killing, their dysregulated expression or activity can adversely impact on the inflammatory process. The existence of efficient endogenous control mechanisms that can dampen or halt this overexuberant protease activity in vivo is essential for the effective resolution of inflammatory lung disease. The function of pulmonary antiproteases is to fulfil this role. Interestingly, in addition to their antiprotease activity, protease inhibitors in the lung also often possess other intrinsic properties that contribute to microbial killing or termination of the inflammatory process. This review will outline important features of chronic inflammation that are regulated by pulmonary proteases and will describe the various mechanisms by which antiproteases attempt to counterbalance exaggerated protease-mediated inflammatory events. These proteases, antiproteases and their modifiers represent interesting targets for therapeutic intervention.

Role of Proteases in Chronic Obstructive Pulmonary Disease

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Kailash C. Pandey

2017-08-01

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

Degradation of tetracycline in aqueous media by ozonation in an internal loop-lift reactor

Energy Technology Data Exchange (ETDEWEB)

Wang Yan [Department of Environmental Engineering, Wuhan University, P.O. Box C319, Luoyu Road 129, Wuhan 430079 (China); Zhang Hui, E-mail: eeng@whu.edu.cn [Department of Environmental Engineering, Wuhan University, P.O. Box C319, Luoyu Road 129, Wuhan 430079 (China); Zhang Jianhua; Lu Chen; Huang Qianqian; Wu Jie; Liu Fang [Department of Environmental Engineering, Wuhan University, P.O. Box C319, Luoyu Road 129, Wuhan 430079 (China)

2011-08-15

The degradation of tetracycline by ozone was investigated in this paper. In the laboratory scale experiments, the effect of major parameters, including pH, gas flow rate, gaseous ozone concentration, hydrogen peroxide concentration and hydroxyl radical scavenger (tert-butyl alcohol) on the degradation of tetracycline was studied. A pseudo-first order kinetic model was used to simulate the experimental results. The results indicated that the tetracycline degradation rate increased with pH, gaseous ozone concentration and gas flow rate. The addition of hydrogen peroxide or hydroxyl radical scavenger had little effect on tetracycline removal, indicating that the direct oxidation of tetracycline by ozone was dominant process and the radical contribution to the tetracycline oxidation could be neglected. The main intermediates were separated and identified as well as the simple degradation pathway of tetracycline was proposed. The COD removal reached to 35% after 90 min reaction. The acute toxicity experiments illustrated that the Daphnia magna mortality reached the maximum after 25 min ozonation and then decreased to zero after 90 min ozonation.

Serine protease inhibitors of parasitic helminths.

Science.gov (United States)

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

2012-05-01

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

Identification of an archaeal presenilin-like intramembrane protease.

Science.gov (United States)

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

2010-09-29

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

The coagulation factor Xa/protease activated receptor-2 axis in the progression of liver fibrosis : a multifaceted paradigm

NARCIS (Netherlands)

Borensztajn, Keren; von der Thusen, Jan H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

2010-01-01

Introduction Activation of the coagulation cascade during liver fibrosis: a puzzling paradox Protease-activated receptors: the link between coagulation cascade activation and liver fibrosis Expression and distribution of human PAR-2 in normal and pathological liver tissue FXa signalling on PAR-2

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

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

2017-11-01

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

Electronic-property dependent interactions between tetracycline and graphene nanomaterials in aqueous solution.

Science.gov (United States)

He, Lin; Liu, Fei-Fei; Zhao, Mengyao; Qi, Zhen; Sun, Xuefei; Afzal, Muhammad Zaheer; Sun, Xiaomin; Li, Yanhui; Hao, Jingcheng; Wang, Shuguang

2018-04-01

Understanding the interactions between graphene nanomaterials (GNMs) and antibiotics in aqueous solution is critical to both the engineering applications of GNMs and the assessment of their potential impact on the fate and transport of antibiotics in the aquatic environment. In this study, adsorption of one common antibiotic, tetracycline, by graphene oxide (GO) and reduced graphene oxide (RGO) was examined with multi-walled carbon nanotubes (MWCNTs) and graphite as comparison. The results showed that the tetracycline adsorption capacity by the four selected carbonaceous materials on the unit mass basis followed an order of GO>RGO>MWCNTs>graphite. Upon normalization by surface area, graphite, RGO and MWCNTs had almost the same high tetracycline adsorption affinity while GO exhibited the lowest. We proposed π-electron-property dependent interaction mechanisms to explain the observed different adsorption behaviors. Density functional theory (DFT) calculations suggested that the oxygen-containing functional groups on GO surface reduced its π-electron-donating ability, and thus decreased the π-based interactions between tetracycline and GO surface. Comparison of adsorption efficiency at different pH indicated that electrostatic interaction also played an important role in tetracycline-GO interactions. Site energy analysis confirmed a highly heterogeneous distribution of the binding sites and strong tetracycline binding affinity of GO surface. Copyright © 2017. Published by Elsevier B.V.

Tetracyclines and tetracycline resistance in agricultural soils: microcosm and field studies.

OpenAIRE

Schmitt, Heike; Stoob, Krispin; Hamscher, Gerd; Smit, Eric; Seinen, Willem

2006-01-01

The influence of the use of antibiotics on the prevalence of resistance genes in the environment is still poorly understood. We studied the diversity of tetracycline and sulfonamide resistance genes as influenced by fertilization with pig manure in soil microcosms and at two field locations. Manure contained a high diversity of resistance genes, regardless of whether it stemmed from a farm operation with low or regular use of antibiotics. In the microcosm soils, the influence of fertilization...

Protease-activated receptor-2 stimulates intestinal epithelial chloride transport through activation of PLC and selective PKC isoforms.

Science.gov (United States)

van der Merwe, Jacques Q; Moreau, France; MacNaughton, Wallace K

2009-06-01

Serine proteases play important physiological roles through their activity at G protein-coupled protease-activated receptors (PARs). We examined the roles that specific phospholipase (PL) C and protein kinase (PK) C (PKC) isoforms play in the regulation of PAR(2)-stimulated chloride secretion in intestinal epithelial cells. Confluent SCBN epithelial monolayers were grown on Snapwell supports and mounted in modified Ussing chambers. Short-circuit current (I(sc)) responses to basolateral application of the selective PAR(2) activating peptide, SLIGRL-NH(2), were monitored as a measure of net electrogenic ion transport caused by PAR(2) activation. SLIGRL-NH(2) induced a transient I(sc) response that was significantly reduced by inhibitors of PLC (U73122), phosphoinositol-PLC (ET-18), phosphatidylcholine-PLC (D609), and phosphatidylinositol 3-kinase (PI3K; LY294002). Immunoblot analysis revealed the phosphorylation of both PLCbeta and PLCgamma following PAR(2) activation. Pretreatment of the cells with inhibitors of PKC (GF 109203X), PKCalpha/betaI (Gö6976), and PKCdelta (rottlerin), but not PKCzeta (selective pseudosubstrate inhibitor), also attenuated this response. Cellular fractionation and immunoblot analysis, as well as confocal immunocytochemistry, revealed increases of PKCbetaI, PKCdelta, and PKCepsilon, but not PKCalpha or PKCzeta, in membrane fractions following PAR(2) activation. Pretreatment of the cells with U73122, ET-18, or D609 inhibited PKC activation. Inhibition of PI3K activity only prevented PKCdelta translocation. Immunoblots revealed that PAR(2) activation induced phosphorylation of both cRaf and ERK1/2 via PKCdelta. Inhibition of PKCbetaI and PI3K had only a partial effect on this response. We conclude that basolateral PAR(2)-induced chloride secretion involves activation of PKCbetaI and PKCdelta via a PLC-dependent mechanism resulting in the stimulation of cRaf and ERK1/2 signaling.

Effect of Tetracycline on Late-stage African trypanosomiasis in Rats ...

African Journals Online (AJOL)

Effect of Tetracycline on Late-stage African trypanosomiasis in Rats. T.O. Johnson, J.T. Ekanem. Abstract. The effect of tetracycline on late stage African trypanosomiasis was examined in an in vivo experiment using rats infected with Trypanosoma brucei brucei. Infected rats were treated on the 5th day of infection with ...

Barley (Hordeum vulgare L.) cysteine proteases: heterologous expression, purification and characterization

DEFF Research Database (Denmark)

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

2011-01-01

During germination of barley seeds, mobilization of protein is essential and cysteine proteases accounts for more than 90 % of the total proteolytic activity in the degradation of barley seed storage proteins. Cysteine proteases exist as pro-enzyme and is activated through reduction of the active...... site cysteines and by removal of the pro-domain. The complement of cysteine proteases is comprehensive and for detailed studies of the individual components of this complement, a fast and efficient eukaryotic expression platform is highly desirable. A cDNA clone of the barley key cysteine endoprotease...

Subacute neuronopathy in a young man: a possible association with tetracycline treatment

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Magnus Vrethem

2011-11-01

Full Text Available A young man with subacute neuronopathy following tetracycline treatment is described. The symptoms started as a sensory dorsal root affection but by time also involved motor nerves. He developed a severe sensory ataxia with pseudoathetotic movements. Other possible aetiologies were scrutinized and excluded. Tetracycline induced neuronopathy is hitherto not reported in the literature. We propose a possible association between treatment with tetracycline and the development of sensory neuronopathy in this patient.

Increasing tetracycline concentrations on the performance and communities of mixed microalgae-bacteria photo-bioreactors

KAUST Repository

Xiong, Yanghui

2017-12-11

This study investigated the impact of varying concentrations of tetracycline on the performance of mixed microalgae-bacteria photo-bioreactors. Photo-bioreactors were assessed for their ability to remove carbon dioxide (CO2) from the biogas of anaerobic membrane bioreactor (anMBR), and nutrients from the anaerobic effluent. The varying concentrations of tetracycline had no impact on the removal of CO2 from biogas. 29% v/v of CO2 was completely removed to generate >20% v/v of oxygen (O2) in all reactors. Removal of nutrients and biomass was not affected at low concentrations of tetracycline (≤150μg/L), but 20mg/L of tetracycline lowered the biomass generation and removal efficiencies of phosphate. Conversely, high chlorophyll a and b content was observed at 20mg/L of tetracycline. High tetracycline level had no impact on the diversity of 18S rRNA gene-based microalgal communities but adversely affected the 16S rRNA gene-based microbial communities. Specifically, both Proteobacteria and Bacteroidetes phyla decreased in relative abundance but not phylum Chloroplast. Additionally, both nitrogen-fixing (e.g. Flavobacterium, unclassified Burkholderiales and unclassified Rhizobiaceae) and denitrifying groups (e.g. Hydrogenophaga spp.) were significantly reduced in relative abundance at high tetracycline concentration. Phosphate-accumulating microorganisms, Acinetobacter spp. and Pseudomonas spp. were similarly reduced upon exposure to high tetracycline concentration. Unclassified Comamonadaceae, however, increased in relative abundance, which correlated with an increase in the abundance of tetracycline resistance genes associated with efflux pump mechanism. Overall, the findings demonstrate that antibiotic concentrations in municipal wastewaters will not significantly affect the removal of nutrients by the mixed microalgae-bacteria photo-bioreactors. However, utilizing such photo-bioreactors as a polishing step for anMBRs that treat wastewaters with high tetracycline

Synergistic Efficacy of Aedes aegypti Antimicrobial Peptide Cecropin A2 and Tetracycline against Pseudomonas aeruginosa

Science.gov (United States)

Zheng, Zhaojun; Tharmalingam, Nagendran; Liu, Qingzhong; Kim, Wooseong; Fuchs, Beth Burgwyn; Zhang, Rijun; Vilcinskas, Andreas

2017-01-01

ABSTRACT The increasing prevalence of antibiotic resistance has created an urgent need for alternative drugs with new mechanisms of action. Antimicrobial peptides (AMPs) are promising candidates that could address the spread of multidrug-resistant bacteria, either alone or in combination with conventional antibiotics. We studied the antimicrobial efficacy and bactericidal mechanism of cecropin A2, a 36-residue α-helical cationic peptide derived from Aedes aegypti cecropin A, focusing on the common pathogen Pseudomonas aeruginosa. The peptide showed little hemolytic activity and toxicity toward mammalian cells, and the MICs against most clinical P. aeruginosa isolates were 32 to 64 μg/ml, and its MICs versus other Gram-negative bacteria were 2 to 32 μg/ml. Importantly, cecropin A2 demonstrated synergistic activity against P. aeruginosa when combined with tetracycline, reducing the MICs of both agents by 8-fold. The combination was also effective in vivo in the P. aeruginosa/Galleria mellonella model (P < 0.001). We found that cecropin A2 bound to P. aeruginosa lipopolysaccharides, permeabilized the membrane, and interacted with the bacterial genomic DNA, thus facilitating the translocation of tetracycline into the cytoplasm. In summary, the combination of cecropin A2 and tetracycline demonstrated synergistic antibacterial activity against P. aeruginosa in vitro and in vivo, offering an alternative approach for the treatment of P. aeruginosa infections. PMID:28483966

Isolation of alkaline protease from Bacillus subtilis AKRS3

African Journals Online (AJOL)

ashok

2012-08-28

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

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

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Fidia Fibriana

2015-09-01

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

Cold denaturation of the HIV-1 protease monomer

DEFF Research Database (Denmark)

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

2017-01-01

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

Tetracycline resistance in semi-arid agricultural soils under long-term swine effluent application.

Science.gov (United States)

Popova, Inna E; Josue, Rosemarie D R; Deng, Shiping; Hattey, Jeffory A

2017-05-04

Annually, millions pounds of antibiotics are released unmetabolized into environment along with animal wastes. Accumulation of antibiotics in soils could potentially induce the persistence of antibiotic resistant bacteria. Antibiotics such as tetracyclines and tetracycline-resistant bacteria have been previously detected in fields fertilized with animal manure. However, little is known about the accumulation of tetracyclines and the development of tetracycline resistance in semi-arid soils. Here we demonstrate that continuous land application with swine effluent, containing trace amounts of chlortetracycline, does not necessarily induce tetracycline resistance in soil bacteria. Based on the testing of more than 3,000 bacteria isolated from the amended soils, we found no significant increase in the occurrence and level of chlortetracycline resistant bacteria in soils after 15 years of continuous swine effluent fertilization. To account for a possible transfer of tetracycline-resistant bacteria originated from the swine effluent to soils, we analyzed two commonly found tetracycline resistant genes, tet(O) and tet(M), in the swine effluent and fertilized soils. Both genes were present in the swine effluent, however, they were not detectable in soils applied with swine effluent. Our data demonstrate that agronomic application of manure from antibiotic treated swine effluent does not necessarily result in the development of antibiotic bacterial resistance in soils. Apparently, concentrations of chlortetracycline present in manure are not significant enough to induce the development of antibiotic bacterial resistance.

Controlled-release of tetracycline and lovastatin by poly(D,L-lactide-co-glycolide acid)-chitosan nanoparticles enhances periodontal regeneration in dogs.

Science.gov (United States)

Lee, Bor-Shiunn; Lee, Chien-Chen; Wang, Yi-Ping; Chen, Hsiao-Jan; Lai, Chern-Hsiung; Hsieh, Wan-Ling; Chen, Yi-Wen

2016-01-01

Chronic periodontitis is characterized by inflammation of periodontal tissues, leading to bone resorption and tooth loss. The goal of treatment is to regenerate periodontal tissues including bone and cementum lost as a consequence of disease. The local delivery of tetracycline was proven to be effective in controlling localized periodontal infection without apparent side effects. Previous studies suggested that lovastatin has a significant role in new bone formation; however, the local delivery of lovastatin might enhance its therapeutic effects. A number of local delivery devices have been developed recently, including poly(D,L-lactide-co-glycolide acid) (PLGA) nanoparticles. The aim of this study was to develop a local delivery device, PLGA-lovastatin-chitosan-tetracycline nanoparticles, which allows the sequential release of tetracycline and lovastatin to effectively control local infection and promote bone regeneration in periodontitis. The size and microstructure of nanoparticles were examined by transmission electron microscopy, Nanoparticle Size Analyzer, and Fourier transform infrared spectroscopy. The release of tetracycline and lovastatin was quantified using a UV-Vis spectrophotometer. Furthermore, the cytotoxic effect and alkaline phosphatase activity of the nanoparticles in osteoblast cell cultures as well as antibacterial activity against periodontal pathogens were investigated. Finally, the bone regeneration potential of PLGA nanoparticles in three-walled defects in beagle dogs was investigated. The results indicated that PLGA-lovastatin-chitosan-tetracycline nanoparticles showed good biocompatibility, antibacterial activity, and increased alkaline phosphatase activity. The volumetric analysis from micro-CT revealed significantly increased new bone formation in defects filled with nanoparticles in dogs. This novel local delivery device might be useful as an adjunctive treatment in periodontal regenerative therapy.

Isolation of microorganisms with chinitase, protease and keratinase activities from petroleum contaminated soils

International Nuclear Information System (INIS)

Cervantes-Gonzalez, E.; Rojas-Avelizapa, L.; Cruz-Camarillo, R.; Rojas-Avelizapa, N.G.

2005-01-01

The most important part in one process of bio-remediation are the microorganisms with the capacities to degrade target compounds, this research is based to find microorganisms hydrocarbon-clastic with enzyme activities to degrade chicken feather (keratinolytic activity) which is also a contaminant and has been used such as sorbent of petroleum and can be composted after the oil spill cleanup is complete, the isolation was also to degrade shrimp waste (chitinolitic and proteolitic activity) which is waste material that can be used in compost or such as sorbent of petroleum too. We isolated mesofilic aerobic microorganisms from mexican soils located in Tabasco, Mexico. We achieved to isolate 105 bacteria from 10 soils, 90% was Bacillus Gram (-) which are common in soils and all were hydrocarbon-clastic, only 7 different bacteria had protease and chitinase activity and 12 bacteria had keratinase activity. So we found three fungi and one actinomycete with capacity to degrade hydrocarbons and presence of chitinase activity. The results of growth and enzyme activities in liquid culture showed that the protease activity was produced between 18 and 48 h in almost all bacteria, the chitinase activity started at 12 h but was slight , only 0.5 U/ml, and the keratinase activity was produced after 6 h of incubation and there were correlation between logarithmic phase of growth and enzymes production. With this study we showed the existence of some enzyme activities from microorganisms that live in hostile habitats. This, can be useful in bio-treatment soils by the possible use of this type of residues that can be bio-degraded at the same time that the hydrocarbons increasing the speed or the quality of cleanup in soils. (authors)

Indispensable Role of Proteases in Plant Innate Immunity.

Science.gov (United States)

Balakireva, Anastasia V; Zamyatnin, Andrey A

2018-02-23

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

Cloned Bacillus subtilis alkaline protease (aprA) gene showing high level of keratinolytic activity.

Science.gov (United States)

Zaghloul, T I

1998-01-01

The Bacillus subtilis alkaline protease(aprA) gene was previously cloned on a pUBHO-derivative plasmid. High levels of expression and gene stability were demonstrated when B. subtilis cells were grown on the laboratory medium 2XSG. B. subtilis cells harboring the multicopy aprA gene were grown on basal medium, supplemented with 1 % chicken feather as a source of energy, carbon, and nitrogen. Proteolytic and keratinolytic activities were monitored throughout the cultivation time. A high level of keratinolytic activity was obtained, and this indicates that alkaline protease is acting as a keratinase. Furthermore, considerable amounts of soluble proteins and free amino acids were obtained as a result of the enzymatic hydrolysis of feather. Biodegradation of feather waste using these cells represents an alternative way to improve the nutritional value of feather, since feather waste is currently utilized on a limited basis as a dietary protein supplement for animal feedstuffs. Moreover, the release of free amino acids from feather and the secreted keratinase enzyme would promote industries based on feather waste.

Identification of an archaeal presenilin-like intramembrane protease.

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Celia Torres-Arancivia

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

Factor VII-activating protease in patients with acute deep venous thrombosis

DEFF Research Database (Denmark)

Sidelmann, Johannes J; Vitzthum, Frank; Funding, Eva

2008-01-01

Factor VII-activating protease (FSAP) is involved in haemostasis and inflammation. FSAP cleaves single chain urokinase-type plasminogen activator (scu-PA). The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene leads to the expression of a FSAP variant with reduced ability to activate scu......-PA, without affecting the ability to activate coagulation Factor VII (FVII). Previous studies have investigated the association of the 1601GA genotype with incidence and progression of carotid stenosis and deep venous thrombosis (DVT). The present study is the first to evaluate the potential association...... between the FSAP phenotype and DVT. We studied the association between the 1601G/A polymorphism, FSAP activity, FSAP antigen, Factor VIIa (FVIIa), prothrombin fragment 1+2 (F1+2), and C-reactive protein (CRP) in plasmas of 170 patients suspected for DVT. FSAP genotypes were equally distributed in patients...

Two Kazal-type protease inhibitors from Macrobrachium nipponense and Eriocheir sinensis: comparative analysis of structure and activities.

Science.gov (United States)

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

2012-03-01

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

ISOLASI DAN KARAKTERISASI PROTEASE ALKALIN DARI ISOLAT BAKTERI LIMBAH TERNAK DI EXFARM FAKULTAS PETERNAKAN UNSOED

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Zusfahair

2011-05-01

Full Text Available Protease is one of the widely used enzymes for the industry. The potential resource of microorganism that produced protease is milk cow waste. In this research, isolation and characterization has been done toward isolated protease from milk cow waste of the Exfarm’s Animal Husbandry Faculty at University of Jenderal Soedirman, Purwokerto. The research used experiment method and the parameters observed were the genus of bacteria which produce protease and the activity of protease. The characterizations of protease were determination of optimum pH and temperature, the influence of metal ions, EDTA, surfactant, and commercial detergent toward enzyme activity, and also the study of enzyme stability. The results from the research showed that the isolated bacteria from the Exfarm’s of Animal Husbandry Faculty of UNSOED, which produced protease was Salmonella sp. Characterization of isolated Salmonella sp. from 45% ammonium sulphate fraction indicated that the optimum temperature was 50 ºC, optimum pH was 8, the enzyme was activated by Ca2+ dan Mg2+ ion, whereas it was inhibited by Zn2+, Cu2+ ions and EDTA. The addition of Tween-80 with the concentration of 0.2% and 0.4% increased protease activity, however the addition of Tween-80 with concentration higher than 0.6% decreased the protease activity. Enzyme protease from isolated Salmonella sp. was relatively stable with the addition of commercial detergent such as Attack, Surf, and Bukrim.

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

NARCIS (Netherlands)

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

2013-01-01

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

Activation of mas-related G-protein-coupled receptors by the house dust mite cysteine protease Der p1 provides a new mechanism linking allergy and inflammation.

Science.gov (United States)

Reddy, Vemuri B; Lerner, Ethan A

2017-10-20

Cysteine and serine proteases function via protease-activated and mas-related G-protein-coupled receptors (Mrgprs) to contribute to allergy and inflammation. Der p1 is a cysteine protease and major allergen from the house dust mite and is associated with allergic rhinitis and allergic asthma. Der p1 activates protease-activated receptor 2 and induces the release of the pro-inflammatory cytokine IL-6 from cells. However, the possibility that Der p1 acts on Mrgprs has not been considered. We report here that ratiometric calcium imaging reveals that Der p1 activates the human receptor MRGPRX1 and the mouse homolog MrgprC11, implicated previously in itch. Der p1 cleavage of N-terminal receptor peptides followed by site-directed mutagenesis of the cleavage sites links receptor activation to specific amino acid residues. Der p1 also induced the release of IL-6 from heterologous cells expressing MRGPRX1. In summary, activation of Mrgprs by the allergen Der p1 may contribute to inflammation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Partial purification and characterization of alkaline proteases from ...

African Journals Online (AJOL)

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

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

Science.gov (United States)

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

2010-10-01

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

Characterization of the Protease Activity of Detergents: Laboratory Practicals for Studying the Protease Profile and Activity of Various Commercial Detergents

Science.gov (United States)

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

2011-01-01

Detergent enzymes account for about 30% of the total worldwide production of enzymes and are one of the largest and most successful applications of modern industrial biotechnology. Proteases can improve the wash performance of household, industrial, and institutional laundry detergents used to remove protein-based stains such as blood, grass, body…

Effect of Tetracycline and Vitamin E on Spatial Memory, Learning, and Depression in Wistar Rats

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Neda Naderi

2015-12-01

Full Text Available Background and Objectives: Tetracyclines are antibiotics that are widely used. Tetracycline, easily passes the blood-brain barrier and protects the nervous system due to its specific chemical structure. Vitamin E is one of the fat-soluble vitamins. This vitamin helps prevent the progression of Alzheimer's disease. The purpose of the present study is to investigate the effects of tetracycline and vitamin E on spatial memory, learning, and depression.   Methods: In this experimental study, 21 male Wistar rats were randomly divided into 3 groups of 7 each. The effect of tetracycline and vitamin E on memory, learning, and depression was investigated using 8-arm radial maze and elevated plus maze. Data were analyzed by one-way ANOVA and Tukey's statistical tests. Statistical significance level was considered p<0.05.   Results: After 21 days of treatment, it was shown that tetracycline antibiotic has a significant effect on memory. Also Vitamin E significantly reduced depression and its protective effect decreased the adverse effect of tetracycline. In the treatment group, vitamin E along with tetracycline had a significant effect on memory loss.   Conclusion: The results of the present study indicated that use of vitamin E can reduce depression by its antioxidant and protective effects. Tetracycline also significantly increases memory. Therefore, according to the results, it is suggested that tetracycline be used along with vitamin E to reduce negative effects of the drug.

Novel inexpensive fungi proteases: Production by solid state fermentation and characterization.

Science.gov (United States)

Novelli, Paula Kern; Barros, Margarida Maria; Fleuri, Luciana Francisco

2016-05-01

A comparative study was carried out for proteases production using agroindustrial residues as substrate for solid state fermentation (SSF) of several fungal strains. High protease production was observed for most of the microorganisms studied, as well as very different biochemical characteristics, including activities at specific temperatures and a wide range of pH values. The enzymes produced were very different regarding optimum pH and they showed stability at 50 °C. Aspergillus oryzae showed stability at all pH values studied. Penicillium roquefortii and Aspergillus flavipes presented optimum activity at temperatures of 50 °C and 90 °C, respectively. Lyophilized protease from A. oryzae reached 1251.60 U/g and yield of 155010.66 U/kg of substrate. Therefore, the substrate as well as the microorganism strain can modify the biochemical character of the enzyme produced. The high protease activity and stability established plus the low cost of substrates, make these fungal proteases potential alternatives for the biotechnological industry. Copyright © 2015 Elsevier Ltd. All rights reserved.

Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

OpenAIRE

Siddiqui, Imran Ali; Haas, Dieter; Heeb, Stephan

2005-01-01

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocon...

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

Science.gov (United States)

Rossano, Rocco; Larocca, Marilena; Riccio, Paolo

2011-09-01

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

Tetracycline-induced renal failure after dental treatment.

Science.gov (United States)

Miller, Craig S; McGarity, Gary J

2009-01-01

Tetracyclines are broad-spectrum antibiotics used by dental practitioners in the treatment of periodontal disease. They generally are safe in adults. However, caution is advised in patients who have pre-existing kidney disease. A 42-year-old woman with polycystic kidney disease received a prescription for tetracycline (250 milligrams, four times daily) after undergoing tooth extractions. She developed nausea, vomiting and diarrhea within days and end-stage renal disease within two weeks of taking the antibiotic. Hemodialysis was required to stabilize the patient's condition. Use of the Naranjo nomogram demonstrated an association between the two events. This case illustrates the importance of obtaining a thorough medical history and understanding potential adverse drug effects before prescribing a common antibiotic.

Ceftriaxone and tetracycline effect on biofilm-formation strains of Staphylococcus epidermidis

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O. I. Sidashenko

2014-04-01

Full Text Available 122 strains of staphylococci were identified. Among the examined 122 clinical strains of staphylococci, 67 strains belonged to coagulase-positive, and 55 strains to the coagulase-negative ones. According to the study of physiological and biochemical properties, it was found that 37 strains (30.3% belonged to S. epidermidis species. One of the biological properties of many bacteria is the ability to film formation and these strains attract special attention, since it is known that the film antibiotic resistance is higher than in planktonic cultures. It was determined that 20 strains of those under study were film-forming, 17 strains – non-biofilm forming ones. The film was formed during three days, and settled to the bottom of the plate holes. The clinical (Cl strain of S. epidermidis was sensitive to ceftriaxone and tetracicline. The control (C strains of S. epidermidis were sensitive to ceftriaxone, tetracycline and sizomicine. The study of biofilm growth for 2, 3 and 4 days of incubation was carried out. The maximum rate of biofilm S. epidermidis C was observed during 2–3 days; there is the most intense increase of cells number from 5.2 × 108 CFU/ml, for S. epidermidis Cl to 5.6 × 108 CFU/ml. The effect of ceftriaxone and tetracycline on biofilm formation by 2 investigation strains of S. epidermidis was found. We determined differences in minimal inhibitory concentrations (MIC for planktonic cultures and biofilm of strains under study. It was established that MIC antibiotics inhibited the growth of planktonic cultures on average 2 times lower compared to the MIC which inhibited the biofilm formation. MIC for planktonic culture of S. epidermidis Cl defined for ceftriaxone was equal to 10 mg/ml, and for tetracycline – 1 mg/ml. MIC of ceftriaxone for the control strain was equal to 12 mg/ml, MIC of tetracycline – 0.7 mg/ml. MIC values for dynamics biofilm formation of S. epidermidis Cl strain on the plater were as follows: to

Effective Degradation of Aqueous Tetracycline Using a Nano-TiO2/Carbon Electrocatalytic Membrane

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Zhimeng Liu

2016-05-01

Full Text Available In this work, an electrocatalytic membrane was prepared to degrade aqueous tetracycline (TC using a carbon membrane coated with nano-TiO2 via a sol-gel process. SEM, XRD, EDS, and XPS were used to characterize the composition and structure of the electrocatalytic membrane. The effect of operating conditions on the removal rate of tetracycline was investigated systematically. The results show that the chemical oxygen demand (COD removal rate increased with increasing residence time while it decreased with increasing the initial concentration of tetracycline. Moreover, pH had little effect on the removal of tetracycline, and the electrocatalytic membrane could effectively remove tetracycline with initial concentration of 50 mg·L−1 (pH, 3.8–9.6. The 100% tetracycline and 87.8% COD removal rate could be achieved under the following operating conditions: tetracycline concentration of 50 mg·L−1, current density of 1 mA·cm−2, temperature of 25 °C, and residence time of 4.4 min. This study provides a new and feasible method for removing antibiotics in water with the synergistic effect of electrocatalytic oxidation and membrane separation. It is evident that there will be a broad market for the application of electrocatalytic membrane in the field of antibiotic wastewater treatment.

A Simple Surface-Enhanced Raman Spectroscopic Method for on-Site Screening of Tetracycline Residue in Whole Milk

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Sagar Dhakal

2018-02-01

Full Text Available Therapeutic and subtherapeutic use of veterinary drugs has increased the risk of residue contamination in animal food products. Antibiotics such as tetracycline are used for mastitis treatment of lactating cows. Milk expressed from treated cows before the withdrawal period has elapsed may contain tetracycline residue. This study developed a simple surface-enhanced Raman spectroscopic (SERS method for on-site screening of tetracycline residue in milk and water. Six batches of silver colloid nanoparticles were prepared for surface enhancement measurement. Milk-tetracycline and water-tetracycline solutions were prepared at seven concentration levels (1000, 500, 100, 10, 1, 0.1, and 0.01 ppm and spiked with silver colloid nanoparticles. A 785 nm Raman spectroscopic system was used for spectral measurement. Tetracycline vibrational modes were observed at 1285, 1317 and 1632 cm−1 in water-tetracycline solutions and 1322 and 1621 cm−1 (shifted from 1317 and 1632 cm−1, respectively in milk-tetracycline solutions. Tetracycline residue concentration as low as 0.01 ppm was detected in both the solutions. The peak intensities at 1285 and 1322 cm−1 were used to estimate the tetracycline concentrations in water and milk with correlation coefficients of 0.92 for water and 0.88 for milk. Results indicate that this SERS method is a potential tool that can be used on-site at field production for qualitative and quantitative detection of tetracycline residues.

A Simple Surface-Enhanced Raman Spectroscopic Method for on-Site Screening of Tetracycline Residue in Whole Milk.

Science.gov (United States)

Dhakal, Sagar; Chao, Kuanglin; Huang, Qing; Kim, Moon; Schmidt, Walter; Qin, Jianwei; Broadhurst, C Leigh

2018-02-01

Therapeutic and subtherapeutic use of veterinary drugs has increased the risk of residue contamination in animal food products. Antibiotics such as tetracycline are used for mastitis treatment of lactating cows. Milk expressed from treated cows before the withdrawal period has elapsed may contain tetracycline residue. This study developed a simple surface-enhanced Raman spectroscopic (SERS) method for on-site screening of tetracycline residue in milk and water. Six batches of silver colloid nanoparticles were prepared for surface enhancement measurement. Milk-tetracycline and water-tetracycline solutions were prepared at seven concentration levels (1000, 500, 100, 10, 1, 0.1, and 0.01 ppm) and spiked with silver colloid nanoparticles. A 785 nm Raman spectroscopic system was used for spectral measurement. Tetracycline vibrational modes were observed at 1285, 1317 and 1632 cm -1 in water-tetracycline solutions and 1322 and 1621 cm -1 (shifted from 1317 and 1632 cm -1 , respectively) in milk-tetracycline solutions. Tetracycline residue concentration as low as 0.01 ppm was detected in both the solutions. The peak intensities at 1285 and 1322 cm -1 were used to estimate the tetracycline concentrations in water and milk with correlation coefficients of 0.92 for water and 0.88 for milk. Results indicate that this SERS method is a potential tool that can be used on-site at field production for qualitative and quantitative detection of tetracycline residues.

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

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Jacky Y Suen

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

Microbial alkaline proteases: Optimization of production parameters and their properties

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Kanupriya Miglani Sharma

2017-06-01

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

Cysteine Protease Inhibitors as Chemotherapy: Lessons from a Parasite Target

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

1999-09-01

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

Analysis of serine proteases from marine sponges by 2-D zymography.

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Wilkesman, Jeff G; Schröder, Heinz C

2007-02-01

Proteolytic activities isolated from the marine demosponges Geodia cydonium and Suberites domuncula were analyzed by 2-D zymography, a technique that combines IEF and zymography. After purification, a 200 kDa proteolytically active protein band was obtained from G. cydonium when analyzed in gelatin copolymerized 1-D zymograms. The enzymatic activity was quantified using alpha-N-benzoyl-D-arginine p-nitroanilide (BAPNA) as a substrate and corresponded to a serine protease. The protease activity was resistant to urea and SDS. DTT and 2-mercaptoethanol (2-ME) did not significantly change the protease activity, but induced a shift in molecular mass of the proteolytic band to lower M(r) values as detected by zymography. Under mild denaturing conditions, lower M(r) bands (zymography, the protease from G. cydonium revealed a pI of 8.0 and an M(r) shift from 200 to 66 kDa. To contrast these results, a cytosolic sample from S. domuncula was analyzed. The proteolytic activity of this sponge after 2-D zymography corresponded to an M(r) of 40 kDa and a pI of 4.0. The biological function of both sponge proteases is not yet known. This study demonstrates that mild denaturing conditions required for IEF may alter the interpretation of the 2-D zymography, and care must be taken during sample preparation.

21 CFR 556.720 - Tetracycline.

Science.gov (United States)

2010-04-01

..., FEEDS, AND RELATED PRODUCTS TOLERANCES FOR RESIDUES OF NEW ANIMAL DRUGS IN FOOD Specific Tolerances for Residues of New Animal Drugs § 556.720 Tetracycline. (a) Acceptable daily intake (ADI). The ADI for total... tissues of calves, swine, sheep, chickens, and turkeys, of 2 parts per million (ppm) in muscle, 6 ppm in...

Structure of the Mature Streptococcal Cysteine Protease Exotoxin mSpeB in Its Active Dimeric Form

DEFF Research Database (Denmark)

Olsen, Johan G; Dagil, Robert; Niclasen, Louise Meinert

2009-01-01

Invasive infections of Streptococcus pyogenes are dependent on the cysteine protease streptococcal pyrogenic exotoxin B. Previous structures of the enzyme have not disclosed the proper active-site configuration. Here, the crystal structure of the mature enzyme is presented to 1.55 A, disclosing...

Study of the behaviour of tetracycline as fission products extracting agent

International Nuclear Information System (INIS)

Cunha, I.I.L.

1983-01-01

Both spectrophotometric and potentiometric titration techniques were used to show the formation of complexes between tetracycline and the elements: zirconium, uranium, molybdenum, strontium, barium and ruthenium. It has been verified that tetracycline does not form complexes with cesium, tellurium and iodine. Those techniques have also been used to determine the sites on the tetracycline molecule at which ions may be bound. The behaviour of tetracycline as an extracting agent for those elements, as well as for niobium and technetium has been studied and the influence of the acidity of the aqueous phase upon extraction of the elements mentioned has been considered. Extraction experiments were carried out in the presence of chloride, perchlorate, nitrate and sulfate ions. Studies have been made to determine whether or not the complex extracted into organic phase is really the complex formed between tetracycline and the elements considered as well as to determine the time of shaking necessary so that the equilibrium between the phases is attained. Based on all information obtained from extraction experiments made for uranium and the fission products Zr-95, Nb-95, Ce-141, La-140, Ru-103, Ba-140 and Cs-137, the possibility of using tetracycline for separating those fission products from each other and from uranium has been studies and a scheme for simultaneous separation of those elements has been proposed. The same study has been made for I-131, Tc-99m, Mo-99, Te-132, Np-239 and uranium. The method described is applicable to the separation of some fission products existing in solutions at tracer levels, and not to be used in nuclear fuel reprocessing or any other industrial application. (Author) [pt

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

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Chen Chaoping

2010-07-01

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

Partial characterisation of digestive proteases of the Mayan cichlid Cichlasoma urophthalmus.

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Cuenca-Soria, C A; Álvarez-González, C A; Ortiz-Galindo, J L; Nolasco-Soria, H; Tovar-Ramírez, D; Guerrero-Zárate, R; Castillo-Domínguez, A; Perera-García, M A; Hernández-Gómez, R; Gisbert, E

2014-06-01

The characterisation of digestive proteases in native freshwater fish such as the Mayan cichlid Cichlasoma urophthalmus provides scientific elements that may be used to design balanced feed that matches with the digestive capacity of the fish. The purpose of this study was to characterise the digestive proteases, including the effect of the pH and the temperature on enzyme activity and stability, as well as the effect of inhibitors using multienzymatic extracts of the stomach and intestine of C. urophthalmus juveniles. Results showed that the optimum activities of the acid and alkaline proteases occurred at pH values of 3 and 9, respectively, whereas their optimum temperatures were 55 and 65 °C, respectively. The acid proteases were most stable at pH values of 2–3 and at temperatures of 35–45 °C, whereas the alkaline proteases were most stable at pH values of 6–9 and at 25–55 °C. The inhibition assays recorded a residual activity of 4% with pepstatin A for the acid proteases. The inhibition of the alkaline proteases was greater than 80% with TPCK, TLCK, EDTA and ovalbumin, and of 60 and 43.8% with PMSF and SBT1, respectively. The results obtained in this study make it possible to state that C. urophthalmus has a sufficiently complete digestive enzyme machinery to degrade food items characteristic of an omnivorous fish species, although specimens showed a tendency to carnivory.

High throughput protease profiling comprehensively defines active site specificity for thrombin and ADAMTS13.

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Kretz, Colin A; Tomberg, Kärt; Van Esbroeck, Alexander; Yee, Andrew; Ginsburg, David

2018-02-12

We have combined random 6 amino acid substrate phage display with high throughput sequencing to comprehensively define the active site specificity of the serine protease thrombin and the metalloprotease ADAMTS13. The substrate motif for thrombin was determined by >6,700 cleaved peptides, and was highly concordant with previous studies. In contrast, ADAMTS13 cleaved only 96 peptides (out of >10 7 sequences), with no apparent consensus motif. However, when the hexapeptide library was substituted into the P3-P3' interval of VWF73, an exosite-engaging substrate of ADAMTS13, 1670 unique peptides were cleaved. ADAMTS13 exhibited a general preference for aliphatic amino acids throughout the P3-P3' interval, except at P2 where Arg was tolerated. The cleaved peptides assembled into a motif dominated by P3 Leu, and bulky aliphatic residues at P1 and P1'. Overall, the P3-P2' amino acid sequence of von Willebrand Factor appears optimally evolved for ADAMTS13 recognition. These data confirm the critical role of exosite engagement for substrates to gain access to the active site of ADAMTS13, and define the substrate recognition motif for ADAMTS13. Combining substrate phage display with high throughput sequencing is a powerful approach for comprehensively defining the active site specificity of proteases.

Evaluation of degradation of antibiotic tetracycline in pig manure by electron beam irradiation.

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Cho, Jae-Young

2010-04-01

This study was carried out to evaluate the degradation efficiency and intermediate products of the tetracycline from artificially contaminated pig manure using of electron beam irradiation as a function of the absorbed dose. The degradation efficiency of tetracycline was 42.77% at 1 kGy, 64.20% at 3 kGy, 77.83% at 5 kGy, and 90.50% at 10 kGy. The initial concentration of tetracycline (300 mg kg(-1)) in pig manure decreased significantly to 24.2 +/- 5.3 mg kg(-1) after electron beam irradiation at 10 kGy. The radiolytic degradation products of tetracycline were 1,4-benzenedicarboxylic acid, hexadecanoic acid, 9-octadecenamide, 11-octadecenamide, and octadecanoic acid.

Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD.

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Lin Qu

2011-09-01

Full Text Available Toll-like receptor 3 (TLR3 and cytosolic RIG-I-like helicases (RIG-I and MDA5 sense viral RNAs and activate innate immune signaling pathways that induce expression of interferon (IFN through specific adaptor proteins, TIR domain-containing adaptor inducing interferon-β (TRIF, and mitochondrial antiviral signaling protein (MAVS, respectively. Previously, we demonstrated that hepatitis A virus (HAV, a unique hepatotropic human picornavirus, disrupts RIG-I/MDA5 signaling by targeting MAVS for cleavage by 3ABC, a precursor of the sole HAV protease, 3C(pro, that is derived by auto-processing of the P3 (3ABCD segment of the viral polyprotein. Here, we show that HAV also disrupts TLR3 signaling, inhibiting poly(I:C-stimulated dimerization of IFN regulatory factor 3 (IRF-3, IRF-3 translocation to the nucleus, and IFN-β promoter activation, by targeting TRIF for degradation by a distinct 3ABCD processing intermediate, the 3CD protease-polymerase precursor. TRIF is proteolytically cleaved by 3CD, but not by the mature 3C(pro protease or the 3ABC precursor that degrades MAVS. 3CD-mediated degradation of TRIF depends on both the cysteine protease activity of 3C(pro and downstream 3D(pol sequence, but not 3D(pol polymerase activity. Cleavage occurs at two non-canonical 3C(pro recognition sequences in TRIF, and involves a hierarchical process in which primary cleavage at Gln-554 is a prerequisite for scission at Gln-190. The results of mutational studies indicate that 3D(pol sequence modulates the substrate specificity of the upstream 3C(pro protease when fused to it in cis in 3CD, allowing 3CD to target cleavage sites not normally recognized by 3C(pro. HAV thus disrupts both RIG-I/MDA5 and TLR3 signaling pathways through cleavage of essential adaptor proteins by two distinct protease precursors derived from the common 3ABCD polyprotein processing intermediate.

Rapid colorimetric sensing of tetracycline antibiotics with in situ growth of gold nanoparticles.

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Shen, Li; Chen, Jing; Li, Na; He, Pingli; Li, Zhen

2014-08-11

A colorimetric assay utilizing the formation of gold nanoparticles was developed to detect tetracycline antibiotics in fluidic samples. Tetracycline antibiotics showed the capability of directly reducing aurate salts into atomic gold which form gold nanoparticles spontaneously under proper conditions. The resulted gold nanoparticles showed characteristic plasmon absorbance at 526 nm, which can be visualized by naked eyes or with a spectrophotometer. UV-vis absorbance of the resulted gold nanoparticles is correlated directly with the concentrations of tetracycline antibiotics in the solution, allowing for quantitative colorimetric detection of tetracycline antibiotics. Reaction conditions, such as pH, temperature, reaction time, and ionic strength were optimized. Sensitivity of the colorimetric assay can be enhanced by the addition of gold nanoparticle seeds, a LOD as low as 20 ng mL(-1) can be achieved with the help of seed particles. The colorimetric assay showed minimum interference from ethanol, methanol, urea, glucose, and other antibiotics such as sulfonamides, amino glycosides etc. Validity of the method was also evaluated on urine samples spiked with tetracycline antibiotics. The method provides a broad spectrum detection method for rapid and sensitive detection of reductive substances such as tetracycline antibiotics in liquid and biological samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Plasma factor VII-activating protease is increased by oral contraceptives and induces factor VII activation in-vivo

DEFF Research Database (Denmark)

Sidelmann, Johannes J; Skouby, Sven O; Kluft, Cornelis

2011-01-01

Oral contraceptive (OC) use influences the hemostatic system significantly and is a risk factor for development of cardiovascular disease. Factor VII-activating protease (FSAP) has potential effects on hemostasis. The 1601GA genotype of the 1601G/A polymorphism in the FSAP gene expresses a FSAP...... progestins. FSAP genotypes, FSAP and factor VII (FVII) plasma measures were assessed at baseline and after 6 cycles of OC. The 1601GA genotype was present in 49 (8.3%) of the women and was associated with significantly reduced levels of FSAP (P=0.001). OC use increased FSAP antigen by 25% and FSAP activity...

Isolation, identification and characterization of organic solvent tolerant protease from Bacillus sp. DAF-01

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Arastoo Badoei-Dalfard

2012-01-01

Full Text Available Introduction: Organic solvent-tolerant bacteria are relatively novel extermophilic microorganisms, which can produce organic tolerant protease with capacity of being used in industrial biotechnology for producing high-value compounds. Therefore, finding of these bacteria has drawn much researchers attention nowadays. Materials and Methods: In this project, samples were collected from a hot spring, located in Jiroft. Samples were incubated in medium supplemented with cyclohexane and toluene for 3 days. Screening of protease producing bacteria was performed on the specific media, SKM (Skim milk agar, based on clear area diameter. The best bacterium was identified based on 16s rDNA gene. Protease activity was considered in different temperatures, pH and organic solvents.Results: Sequence alignment and phylogenetic tree results showed that this bacteria was closely related to Bacillus niacini, with 97% homology. Enzymatic studies showed that, this enzyme was active at a wide range of temperatures, 20-90 °C and it,s optimal activity was in 60 °C. In addition, maximum protease activity was obtained in the 8-9 range of pH, and optimal stability was also at pH 9.0. Protease activity in the presence of methanol, toluene, isopropanol, cyclohexane and DMF ‏showed that, remaining activity was at least 80% compared to the control (without organic solvent Discussion and Conclusion: Thermopilic capacity, being active in alkaline protease and high protease stability in the presence of organic solvents all herald a remarkable application for using in different industries.

Jojoba seed meal proteins associated with proteolytic and protease inhibitory activities.

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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.

Extracellular Protease of Pseudomonas fluorescens CHA0, a Biocontrol Factor with Activity against the Root-Knot Nematode Meloidogyne incognita

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Siddiqui, Imran Ali; Haas, Dieter; Heeb, Stephan

2005-01-01

In Pseudomonas fluorescens CHA0, mutation of the GacA-controlled aprA gene (encoding the major extracellular protease) or the gacA regulatory gene resulted in reduced biocontrol activity against the root-knot nematode Meloidogyne incognita during tomato and soybean infection. Culture supernatants of strain CHA0 inhibited egg hatching and induced mortality of M. incognita juveniles more strongly than did supernatants of aprA and gacA mutants, suggesting that AprA protease contributes to biocontrol. PMID:16151170

Human eosinophils constitutively express a unique serine protease, PRSS33.

Science.gov (United States)

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

2017-07-01

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

Inhibition of dengue virus replication by novel inhibitors of RNA-dependent RNA polymerase and protease activities.

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Pelliccia, Sveva; Wu, Yu-Hsuan; Coluccia, Antonio; La Regina, Giuseppe; Tseng, Chin-Kai; Famiglini, Valeria; Masci, Domiziana; Hiscott, John; Lee, Jin-Ching; Silvestri, Romano

2017-12-01

Dengue virus (DENV) is the leading mosquito-transmitted viral infection in the world. With more than 390 million new infections annually, and up to 1 million clinical cases with severe disease manifestations, there continues to be a need to develop new antiviral agents against dengue infection. In addition, there is no approved anti-DENV agents for treating DENV-infected patients. In the present study, we identified new compounds with anti-DENV replication activity by targeting viral replication enzymes - NS5, RNA-dependent RNA polymerase (RdRp) and NS3 protease, using cell-based reporter assay. Subsequently, we performed an enzyme-based assay to clarify the action of these compounds against DENV RdRp or NS3 protease activity. Moreover, these compounds exhibited anti-DENV activity in vivo in the ICR-suckling DENV-infected mouse model. Combination drug treatment exhibited a synergistic inhibition of DENV replication. These results describe novel prototypical small anti-DENV molecules for further development through compound modification and provide potential antivirals for treating DENV infection and DENV-related diseases.

Production and partial characterization of alkaline protease from bacillus subtilis mutant induced by gamma radiation

International Nuclear Information System (INIS)

Ibrahim, H.M.M.; Bashandy, A.S.

2010-01-01

Fourteen bacterial isolates belonging to B.subtilis were locally isolated from soil and screened for alkaline protease production. Only one strain, the highly potent one, was selected as alkaline protease producer and subjected to further studies to optimize its production. Alkaline protease production was maximum at 35 degree C after 72 h of incubation and at ph 10.0. molasses as a carbon source and combination of peptone and yeast extract as a nitrogen source enhanced greatly alkaline protease production. The mutant strain induced by gamma radiation showed higher alkaline protease production by 1.97 fold as compared with the parent strain. The alkaline protease enzyme was active at 40 degree C and ph 10. It was compatible with many commercial detergents and showed high stability (84 %) of its original activity with Ariel detergent. Moreover, alkaline protease enhanced the washing performance, and retained 95 % of its activity in the formulated dry powder.

Purification and characterisation of a salt-stable protease from the halophilic archaeon Halogranum rubrum.

Science.gov (United States)

Gao, Ruichang; Shi, Tong; Liu, Xiangdong; Zhao, Mengqin; Cui, Henglin; Yuan, Li

2017-03-01

Because proteases play an important role in the fermentation of fish sauce, the purification and characterisation of an extracellular protease from the halophilic archaeon Halogranum rubrum was investigated. The molecular mass of the protease was estimated to be approximately 47 kDa based on sodium dodecyl sulfate-polyacrylamide gel electropheresis (SDS-PAGE) and native-PAGE analysis. The optimum conditions for catalytic activity were pH 8.0 and 50°C. The protease showed alkaline stability (pH 7.0-10.0). The protease also exhibited novel catalytic ability over a broad range of salinity (NaCl 0-3 mol L -1 ). Calcium ion enhanced the proteolytic activity of the enzyme. The K m and V max values of the purified protease for casein were calculated to be 4.89 mg mL -1 and 1111.11 U mL -1 , respectively. The protease was strongly inhibited by ethylenediamine tetraacetic acid (EDTA) and phenylmethanesulfonyl fluoride (PMSF). Meanwhile, the protease was stable in the presence of Triton X-100, isopropanol, ethanol or dithio-bis-nitrobenzoic (DTNB), but was inhibited by sodium dodecyl sulfate (SDS), dimethyl sulfoxide (DMSO) or methanol. MALDI -TOF/TOF MS analysis revealed that the protease shared some functional traits with protease produced by Halogranum salarium. Furthermore, it exhibited high hydrolytic activity on silver carp myosin protein. The protease is an alkaline and salt-tolerant enzyme that hydrolyses silver carp myosin with high efficiency. These excellent characteristics make this protease an attractive candidate for industrial use in low-salt fish sauce fermentation. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

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

Science.gov (United States)

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

2014-08-01

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

[Hygienic substantiation of the permissible levels for tetracycline-group antibiotics in food].

Science.gov (United States)

Onishchenko, G G; Sheveleva, S A; Khotimchenko, S A

2012-01-01

For the purpose of justification of the hygienic standard for tetracycline-group antibiotics in the food production established in the Russian Federation at more rigid level, than maximum and admissible levels (MAL) of the Codex Alimentarius Commission, the analysis of data of literature on negative nature of impact of low concentration of these antibiotics on an organism and the environmental conditions and risk for health has been performed. Inadequacy of the accepted admissible daily dose (ADD) accepted by The Joint FAO/WHO Expert Committee on Food Additives (JECFA) on action on selection of resistant E. coli in intestines, for the wide contingent of consumers in connection with ignoring of obvious factors of uncertainty (gastrointestinal dysbiosis, age and individual variations in the microbiota of people synergy with other antibiotics residues in food and indirect impact on an organism through microflora from the natural habitat (resistance genes, modified causative organisms with altered properties).. By the analysis of information received with the use of modern molecular and genetic methods, the role of Subinhibitory concentrations (sub-MICs) of tetracyclines as biologically active substances, signaling molecules which, without causing obvious negative consequences in a macroorganism, serve as a major factor of regulation of a transcription in microorganisms and activation of a horizontal gene transfer coding resistance, transferred on conjugative transposons of Tn916-Tn1545 family. Reasonable scientific data on a dominating contribution of minor levels of tetracyclines in globalization in the nature of the most adverse transmissive type of the antibiotic resistance interfaced to formation new bacterial pathotypes, as consequences of irrationally high scales of application in agriculture and strengthened impact on microbic ecosystems of live organisms and objects of habitat are presented. For minimization of this mediated risk for health the need of

Purification and biochemical characterization of the haloalkaliphilic archaeon Natronococcus occultus extracellular serine protease

DEFF Research Database (Denmark)

Studdert, C A; Herrera Seitz, M K; Plasencia, I

2001-01-01

A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.5-12), is rat......A serine protease was purified from Natronococcus occultus stationary phase culture medium (328-fold, yield 19%) and characterized at the biochemical level. The enzyme has a native molecular mass of 130 kDa, has chymotrypsin-like activity, is stable and active in a broad pH range (5.......5-12), is rather thermophilic (optimal activity at 60 degrees C in 1-2 M NaCl) and is dependent on high salt concentrations for activity and stability (1-2 M NaCl or KCl). Polyclonal antibodies were raised against the purified protease. In Western blots, they presented no cross-reactivity with culture medium from...... other halobacteria nor with commercial proteases except subtilisin. The amino acid sequences of three tryptic peptides obtained from Natronococcus occultus protease did not show significant similarity to other known proteolytic enzymes. This fact, in addition to its high molecular mass suggests...

Isolation of protease producing novel Bacillus cereus and detection ...

African Journals Online (AJOL)

user

2011-02-14

Feb 14, 2011 ... The highest protease activity was determined at 30°C temperature and 6.4 pH conditions and after the 18th hour, it decreased evidently. Key words: Protease, production, optimization, Bacillus sp. INTRODUCTION. Enzymes have been produced in large industrial scale for several decades (Falch, 1991).

Inactivation of brain mitochondrial Lon protease by peroxynitrite precedes electron transport chain dysfunction.

Science.gov (United States)

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.

Detection of protease activity by fluorescent protein FRET sensors: from computer simulation to live cells

Science.gov (United States)

Goryashchenko, Alexander S.; Khrenova, Maria G.; Savitsky, Alexander P.

2018-04-01

Förster resonance energy transfer (FRET) sensors are widely used for the detection of protease activity in vitro and in vivo. Usually they consist of a FRET pair connected with a polypeptide linker containing a specific cleavage site for the relevant protease. Use of the fluorescent proteins as components of the FRET pair allows genetic encoding of such sensors and solves the problem of their delivery into live cells and animals. There are several ways to improve the properties of such sensors, mainly to increase FRET efficiency and therefore the dynamic range. One of the ways to achieve this is to use a non-fluorescent chromoprotein as an acceptor. Molecular dynamic simulations may assist in the construction of linker structures connecting donor and acceptor molecules. Estimation of the orientation factor κ 2 can be obtained by methods based on quantum theory and combined quantum mechanics/molecular mechanics approaches. The linker can be structured by hydrophobic interactions, bringing it into a closed conformation that shortens the distance between donor and acceptor and, consequently, increases FRET efficiency. We analyzed the effects of different linker structures on the detection of caspase-3 activity using a non-fluorescent acceptor. Also we have constructed the Tb3+- TagRFP sensor in which a complex of the terbium ion and terbium-binding peptide is used as a donor. This allowed us to use the unique property of lanthanide ions—fluorescence lifetime up to milliseconds—to perform measurements with time delay and exclude the nanosecond-order fluorescence. Using our systems as a starting point, by changing the recognition site in the linker it is possible to perform imaging of different protease activity in vitro or in vivo.

Controlled-release of tetracycline and lovastatin by poly(D,L-lactide-co-glycolide acid-chitosan nanoparticles enhances periodontal regeneration in dogs

Directory of Open Access Journals (Sweden)

Lee BS

2016-01-01

Full Text Available Bor-Shiunn Lee,1 Chien-Chen Lee,2 Yi-Ping Wang,2 Hsiao-Jan Chen,3 Chern-Hsiung Lai,4 Wan-Ling Hsieh,1 Yi-Wen Chen2 1Graduate Institute of Oral Biology, 2Graduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University and National Taiwan University Hospital, 3Department of Clinical Laboratory Sciences and Medical Biotechnology, College of Medicine, National Taiwan University, Taipei, 4College of Life Science, Kaohsiung Medical University, Kaohsiung, Taiwan Abstract: Chronic periodontitis is characterized by inflammation of periodontal tissues, leading to bone resorption and tooth loss. The goal of treatment is to regenerate periodontal tissues including bone and cementum lost as a consequence of disease. The local delivery of tetracycline was proven to be effective in controlling localized periodontal infection without apparent side effects. Previous studies suggested that lovastatin has a significant role in new bone formation; however, the local delivery of lovastatin might enhance its therapeutic effects. A number of local delivery devices have been developed recently, including poly(D,L-lactide-co-glycolide acid (PLGA nanoparticles. The aim of this study was to develop a local delivery device, PLGA-lovastatin-chitosan-tetracycline nanoparticles, which allows the sequential release of tetracycline and lovastatin to effectively control local infection and promote bone regeneration in periodontitis. The size and microstructure of nanoparticles were examined by transmission electron microscopy, Nanoparticle Size Analyzer, and Fourier transform infrared spectroscopy. The release of tetracycline and lovastatin was quantified using a UV-Vis spectrophotometer. Furthermore, the cytotoxic effect and alkaline phosphatase activity of the nanoparticles in osteoblast cell cultures as well as antibacterial activity against periodontal pathogens were investigated. Finally, the bone regeneration potential of PLGA nanoparticles in

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

Science.gov (United States)

Nishiyama, Katsuhiko

2017-08-01

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

Nucleic Acid Aptamers Against Proteases

DEFF Research Database (Denmark)

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

2011-01-01

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

Vorapaxar: The Current Role and Future Directions of a Novel Protease-Activated Receptor Antagonist for Risk Reduction in Atherosclerotic Disease.

Science.gov (United States)

Gryka, Rebecca J; Buckley, Leo F; Anderson, Sarah M

2017-03-01

Despite the current standard of care, patients with cardiovascular disease remain at a high risk for recurrent events. Inhibition of thrombin-mediated platelet activation through protease-activated receptor-1 antagonism may provide reductions in atherosclerotic disease beyond those achievable with the current standard of care. Our primary objective is to evaluate the clinical literature regarding the role of vorapaxar (Zontivity™) in the reduction of cardiovascular events in patients with a history of myocardial infarction and peripheral artery disease. In particular, we focus on the potential future directions for protease-activating receptor antagonists in the treatment of a broad range of atherosclerotic diseases. A literature search of PubMed and EBSCO was conducted to identify randomized clinical trials from August 2005 to June 2016 using the search terms: 'vorapaxar', 'SCH 530348', 'protease-activated receptor-1 antagonist', and 'Zontivity™'. Bibliographies were searched and additional resources were obtained. Vorapaxar is a first-in-class, protease-activated receptor-1 antagonist. The Thrombin Receptor Antagonist for Clinical Event Reduction (TRACER) trial did not demonstrate a significant reduction in a broad primary composite endpoint. However, the Thrombin-Receptor Antagonist in Secondary Prevention of Atherothrombotic Ischemic Events (TRA 2°P-TIMI 50) trial examined a more traditional composite endpoint and found a significant benefit with vorapaxar. Vorapaxar significantly increased bleeding compared with standard care. Ongoing trials will help define the role of vorapaxar in patients with peripheral arterial disease, patients with diabetes mellitus, and other important subgroups. The use of multivariate modeling may enable the identification of subgroups with maximal benefit and minimal harm from vorapaxar. Vorapaxar provides clinicians with a novel mechanism of action to further reduce the burden of ischemic heart disease. Identification of

Tetracyclines Disturb Mitochondrial Function across Eukaryotic Models: A Call for Caution in Biomedical Research

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Norman Moullan

2015-03-01

Full Text Available In recent years, tetracyclines, such as doxycycline, have become broadly used to control gene expression by virtue of the Tet-on/Tet-off systems. However, the wide range of direct effects of tetracycline use has not been fully appreciated. We show here that these antibiotics induce a mitonuclear protein imbalance through their effects on mitochondrial translation, an effect that likely reflects the evolutionary relationship between mitochondria and proteobacteria. Even at low concentrations, tetracyclines induce mitochondrial proteotoxic stress, leading to changes in nuclear gene expression and altered mitochondrial dynamics and function in commonly used cell types, as well as worms, flies, mice, and plants. Given that tetracyclines are so widely applied in research, scientists should be aware of their potentially confounding effects on experimental results. Furthermore, these results caution against extensive use of tetracyclines in livestock due to potential downstream impacts on the environment and human health.

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

Science.gov (United States)

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

2007-11-18

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

Contribution to the study of calcium metabolism in rats treated with tetracycline

International Nuclear Information System (INIS)

Goncalves, M.R.B.

1980-01-01

The tetracycline is one of the most used antibiotics. The interferences in the rats calcium metabolism were studied. Sixteen rats, R dutch type were treated with a 1 mg/100 g of corporal weight, of tetracycline twice a day, for 23 days. In the twentieth day of the treatment, a dose of Calcium 45 was administrated to verify thhe decay curve of the radionuclide plasmatic concentration. A control group of 16 rats was studied to compare the results. A significative decrease of the calcemy and of bone reabsorption in the group treated with tetracycline were observed. (L.M.J.)

Synthesis of glycinamides using protease immobilized magnetic nanoparticles

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Abha Sahu

2016-12-01

Full Text Available In the present investigation, Bacillus subtilis was isolated from slaughterhouse waste and screened for the production of protease enzyme. The purified protease was successfully immobilized on magnetic nanoparticles (MNPs and used for the synthesis of series of glycinamides. The binding and thermal stability of protease on MNPs was confirmed by FTIR spectroscopy and TGA analysis. The surface morphology of MNPs before and after protease immobilization was carried out using SEM analysis. XRD pattern revealed no phase change in MNPs after enzyme immobilization. The processing parameters for glycinamides synthesis viz. temperature, pH, and time were optimized using Response Surface Methodology (RSM by using Design Expert (9.0.6.2. The maximum yield of various amides 2 butyramidoacetic acid (AMD-1,83.4%, 2-benzamidoacetic acid (AMD-2,80.5% and 2,2′((carboxymethyl amino-2-oxoethyl-2-hydroxysuccinylbis(azanediyldiacetic acid (AMD-3,80.8% formed was observed at pH-8, 50 °C and 30 min. The synthesized immobilized protease retained 70% of the initial activity even after 8 cycles of reuse.

BIOCHEMICAL AND HISTOLOGICAL EFFECTS OF TETRACYCLINES ON SPONTANEOUS OSTEOARTHRITIS IN GUINEA PIGS

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Edin De Bri

2011-05-01

Full Text Available Matrix metalloproteinases (MMPs are mediators in connective tissue destruction in a variety of pathologic processes. Recently discovered chemically modified tetracyclines have been found to be effective inhibitors of MMP mediated connective tissue degradation in both rheumatoid arthritis (RA and osteoarthritis (OA. The Hartley guinea pig model has been described with a high incidence of spontaneous OA-like changes in the knee joint. Therefore we have studied the effect of two tetracyclines, doxycycline (Dox and chemically modified tetracycline-7 (CMT-7 which have both previously been shown as potent MMP inhibitors. We found that prophylactic orally given CMT-7 decreases OA changes in the knee joints both in vitro and in vivo in the guinea pig OA model. OA changes were most severe in the central compartment of the medial condyle in the control group. Cartilage fibrillation and destruction, in addition to subchondral bone sclerosis and cyst formation were all less in the CMT-7 treated group compared with controls. Collagen, hyaluronan and proteoglycan content in cartilage was higher in the CMT-7 treated group compared with controls. In contrast, OA changes were not decreased in the Dox group. These results show that tetracyclines, but not all tetracyclines, can reduce the severity of OA in the guinea pig model of spontaneous OA.

Interdependence of Inhibitor Recognition in HIV-1 Protease.

Science.gov (United States)

Paulsen, Janet L; Leidner, Florian; Ragland, Debra A; Kurt Yilmaz, Nese; Schiffer, Celia A

2017-05-09

Molecular recognition is a highly interdependent process. Subsite couplings within the active site of proteases are most often revealed through conditional amino acid preferences in substrate recognition. However, the potential effect of these couplings on inhibition and thus inhibitor design is largely unexplored. The present study examines the interdependency of subsites in HIV-1 protease using a focused library of protease inhibitors, to aid in future inhibitor design. Previously a series of darunavir (DRV) analogs was designed to systematically probe the S1' and S2' subsites. Co-crystal structures of these analogs with HIV-1 protease provide the ideal opportunity to probe subsite interdependency. All-atom molecular dynamics simulations starting from these structures were performed and systematically analyzed in terms of atomic fluctuations, intermolecular interactions, and water structure. These analyses reveal that the S1' subsite highly influences other subsites: the extension of the hydrophobic P1' moiety results in 1) reduced van der Waals contacts in the P2' subsite, 2) more variability in the hydrogen bond frequencies with catalytic residues and the flap water, and 3) changes in the occupancy of conserved water sites both proximal and distal to the active site. In addition, one of the monomers in this homodimeric enzyme has atomic fluctuations more highly correlated with DRV than the other monomer. These relationships intricately link the HIV-1 protease subsites and are critical to understanding molecular recognition and inhibitor binding. More broadly, the interdependency of subsite recognition within an active site requires consideration in the selection of chemical moieties in drug design; this strategy is in contrast to what is traditionally done with independent optimization of chemical moieties of an inhibitor.