A new selective substrate for cathepsin E based on the cleavage site sequence of α2-macroglobulin

2005 ◽  
Vol 386 (3) ◽  
pp. 299-305 ◽  
Author(s):  
Yoshiyuki Yasuda ◽  
Keiko Kohmura ◽  
Tomoko Kadowaki ◽  
Takayuki Tsukuba ◽  
Kenji Yamamoto
Keyword(s):  
Cleavage Site ◽  
Defense Mechanisms ◽  
Enzyme Concentration ◽  
Cysteine Proteinases ◽  
Peptide Substrate ◽  
Enzyme Preparations ◽  
Cathepsin E ◽  
Α2 Macroglobulin ◽  
Highly Sensitive

Abstract Cathepsin E is an intracellular aspartic proteinase of the pepsin family predominantly expressed in cells of the immune system and believed to contribute to homeostasis by participating in host defense mechanisms. Studies on its enzymatic properties, however, have been limited by a lack of sensitive and selective substrates. For a better understanding of the importance of this enzyme in vivo, we designed and synthesized a highly sensitive peptide substrate for cathepsin E based on the sequence of the specific cleavage site of α2-macroglobulin. The substrate constructed, MOCAc-Gly-Ser-Pro-Ala-Phe-Leu-Ala-Lys(Dnp)-D-Arg-NH2[where MOCAc is (7-methoxycoumarin-4-yl)acetyl and Dnp is dinitrophenyl], derived from the cleavage site sequence of human α2-macroglobulin, was the most sensitive and selective for cathepsin E, with k cat/K m values of 8–11 μM-1 S-1, whereas it was resistant to hydrolysis by the analogous aspartic proteinases cathepsin D and pepsin, as well as the lysosomal cysteine proteinases cathepsins B, L, and H. The assay allows the detection of a few fmol of cathepsin E, even in the presence of plasma and cell lysate, and gives accurate results over a wide enzyme concentration range. This substrate might represent a useful tool for monitoring and accurately quantifying cathepsin E, even in crude enzyme preparations.

scholarly journals Inhibition of aspartic proteinases by α2-macroglobulin

1989 ◽  
Vol 259 (3) ◽  
pp. 905-907 ◽  
Author(s):  
D J Thomas ◽  
A D Richards ◽  
J Kay
Keyword(s):  
Cathepsin D ◽  
Serine Proteinases ◽  
Cysteine Proteinases ◽  
Aspartic Proteinases ◽  
Protein Substrates ◽  
Cathepsin E ◽  
Α2 Macroglobulin

The effect of alpha 2-macroglobulin, one of the major antiproteinases in the plasma of vertebrates, on the action of the aspartic proteinases chymosin, cathepsin D and cathepsin E towards peptide and protein substrates at pH 6.2 was examined. Activities towards protein substrates were blocked, thus demonstrating that alpha 2-macroglobulin can inhibit aspartic proteinases, in addition to serine proteinases, cysteine proteinases and metalloproteinases.

Synthesis and 31 P NMR characterization of new low toxic highly sensitive pH probes designed for in vivo acidic pH studies

2002 ◽  
Vol 10 (5) ◽  
pp. 1451-1458 ◽  
Author(s):  
Sophie Martel ◽  
Jean-Louis Clément ◽  
Agnès Muller ◽  
Marcel Culcasi ◽  
Sylvia Pietri
Keyword(s):  
Acidic Ph ◽  
Highly Sensitive ◽  
Nmr Characterization ◽  
Low Toxic ◽  
Ph Probes

scholarly journals The Structure–Properties–Cytotoxicity Interplay: A Crucial Pathway to Determining Graphene Oxide Biocompatibility

2021 ◽  
Vol 22 (10) ◽  
pp. 5401
Author(s):  
Marta Dziewięcka ◽  
Mirosława Pawlyta ◽  
Łukasz Majchrzycki ◽  
Katarzyna Balin ◽  
Sylwia Barteczko ◽  
...  
Keyword(s):  
Graphene Oxide ◽  
Defense Mechanisms ◽  
Physicochemical Analysis ◽  
Experimental Models ◽  
Acheta Domesticus ◽  
Synthesis Process ◽  
Synthesis Methods ◽  
Toxicity Potential ◽  
Potential Applications

Interest in graphene oxide nature and potential applications (especially nanocarriers) has resulted in numerous studies, but the results do not lead to clear conclusions. In this paper, graphene oxide is obtained by multiple synthesis methods and generally characterized. The mechanism of GO interaction with the organism is hard to summarize due to its high chemical activity and variability during the synthesis process and in biological buffers’ environments. When assessing the biocompatibility of GO, it is necessary to take into account many factors derived from nanoparticles (structure, morphology, chemical composition) and the organism (species, defense mechanisms, adaptation). This research aims to determine and compare the in vivo toxicity potential of GO samples from various manufacturers. Each GO sample is analyzed in two concentrations and applied with food. The physiological reactions of an easy model Acheta domesticus (cell viability, apoptosis, oxidative defense, DNA damage) during ten-day lasting exposure were observed. This study emphasizes the variability of the GO nature and complements the biocompatibility aspect, especially in the context of various GO-based experimental models. Changes in the cell biomarkers are discussed in light of detailed physicochemical analysis.

Optically pumped magnetometers enable a new level of biomagnetic measurements

2020 ◽  
Vol 9 (5) ◽  
pp. 247-251
Author(s):  
Tilmann Sander ◽  
Anna Jodko-Władzińska ◽  
Stefan Hartwig ◽  
Rüdiger Brühl ◽  
Thomas Middelmann
Keyword(s):  
Quantum Interference ◽  
High Sensitivity ◽  
High Temporal Resolution ◽  
Optically Pumped ◽  
New Class ◽  
Magnetic Shields ◽  
Highly Sensitive ◽  
Electric And Magnetic Fields ◽  
Auditory Evoked Fields

AbstractThe electrophysiological activities in the human body generate electric and magnetic fields that can be measured noninvasively by electrodes on the skin, or even, not requiring any contact, by magnetometers. This includes the measurement of electrical activity of brain, heart, muscles and nerves that can be measured in vivo and allows to analyze functional processes with high temporal resolution. To measure these extremely small magnetic biosignals, traditionally highly sensitive superconducting quantum-interference devices have been used, together with advanced magnetic shields. Recently, they have been complemented in usability by a new class of sensors, optically pumped magnetometers (OPMs). These quantum sensors offer a high sensitivity without requiring cryogenic temperatures, allowing the design of small and flexible sensors for clinical applications. In this letter, we describe the advantages of these upcoming OPMs in two exemplary applications that were recently carried out at Physikalisch-Technische Bundesanstalt (PTB): (1) magnetocardiography (MCG) recorded during exercise and (2) auditory-evoked fields registered by magnetoencephalography.

Chromogenic Peptide Substrate Assays for Plasma Inhibitors of Plasma Kallikrein: Identification of the Major Inhibitors

1979 ◽  
Author(s):  
M.J. Gallimore ◽  
E. Amundsen ◽  
M. Larsbraaten ◽  
K. Lyngaas ◽  
E. Fareid
Keyword(s):  
Plasma Concentrations ◽  
Gel Filtration ◽  
Plasma Kallikrein ◽  
Peptide Substrate ◽  
Total Inhibition ◽  
Α2 Macroglobulin ◽  
Dependent Inhibition ◽  
Plus Fraction ◽  
Time Dependent Inhibition ◽  
Esterase Inhibitor

Plasma inhibitors of plasma kallikrein(KK) were studied using chromogenic peptide substrate assays. Both “immediate” and “time-dependent” inhibition was detected. Sephadex G-150 gel filtration revealed that fractions containing α2-macroglobulin (α2 M), C1 - esterase inhibitor (CIINH) and a low molecular weight component(KKI3) gave “immediate” inhibition. When fractions were tested for “total” inhibition (incubation of enzyme plus fraction for 300 seconds at 37°C) CIINH was found to be the major inhibitor. Both the α2M and KKI3-containing fractions exhibited more inhibition than in the “immediate” inhibition assay. Studies with purified preparations of CIINH and α2 M indicated that these are the two most important plasma inhibitors of KK. Preparations of α1-antitrypsin (α1AT), antithrombin III (ATIII) and α2-antiplasmin (α2AP) produced insignificant inhibition. When “total” KK inhibition in plasma samples from 20 healthy subjects was compared with plasma concentrations of CIINH, α2M and α1AT (immunochemical assays) a very good correlation (r=0.81) was found between percentage inhibition and CIINH concentration. Correlation values for the other antiproteases were α2M r=0.36 and α1AT r=0.19.

scholarly journals Real-Time In Vivo Bioluminescent Imaging for Evaluating the Efficacy of Antibiotics in a Rat Staphylococcus aureus Endocarditis Model

2005 ◽  
Vol 49 (1) ◽  
pp. 380-387 ◽  
Author(s):  
Yan Q. Xiong ◽  
Julie Willard ◽  
Jagath L. Kadurugamuwa ◽  
Jun Yu ◽  
Kevin P. Francis ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Real Time ◽  
Imaging System ◽  
Bioluminescent Imaging ◽  
Vancomycin Therapy ◽  
Treatment Groups ◽  
Highly Sensitive ◽  
Relevant Animal Model ◽  
And Control

ABSTRACT Therapeutic options for invasive Staphylococcus aureus infections have become limited due to rising antimicrobial resistance, making relevant animal model testing of new candidate agents more crucial than ever. In the present studies, a rat model of aortic infective endocarditis (IE) caused by a bioluminescently engineered, biofilm-positive S. aureus strain was used to evaluate real-time antibiotic efficacy directly. This strain was vancomycin and cefazolin susceptible but gentamicin resistant. Bioluminescence was detected and quantified daily in antibiotic-treated and control animals with IE, using a highly sensitive in vivo imaging system (IVIS). Persistent and increasing cardiac bioluminescent signals (BLS) were observed in untreated animals. Three days of vancomycin therapy caused significant reductions in both cardiac BLS (>10-fold versus control) and S. aureus densities in cardiac vegetations (P < 0.005 versus control). However, 3 days after discontinuation of vancomycin therapy, a greater than threefold increase in cardiac BLS was observed, indicating relapsing IE (which was confirmed by quantitative culture). Cefazolin resulted in modest decreases in cardiac BLS and bacterial densities. These microbiologic and cardiac BLS differences during therapy correlated with a longer time-above-MIC for vancomycin (>12 h) than for cefazolin (∼4 h). Gentamicin caused neither a reduction in cardiac S. aureus densities nor a reduction in BLS. There were significant correlations between cardiac BLS and S. aureus densities in vegetations in all treatment groups. These data suggest that bioluminescent imaging provides a substantial advance in the real-time monitoring of the efficacy of therapy of invasive S. aureus infections in live animals.

scholarly journals Ophthalmic Drug Dosage Forms: Characterisation and Research Methods

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Przemysław Baranowski ◽  
Bożena Karolewicz ◽  
Maciej Gajda ◽  
Janusz Pluta
Keyword(s):  
Defense Mechanisms ◽  
Drug Application ◽  
Drug Dosage ◽  
Eye Drops ◽  
Eye Tissues ◽  
Ophthalmic Drug ◽  
Application Frequency

This paper describes hitherto developed drug forms for topical ocular administration, that is, eye drops, ointments,in situgels, inserts, multicompartment drug delivery systems, and ophthalmic drug forms with bioadhesive properties. Heretofore, many studies have demonstrated that new and more complex ophthalmic drug forms exhibit advantage over traditional ones and are able to increase the bioavailability of the active substance by, among others, reducing the susceptibility of drug forms to defense mechanisms of the human eye, extending contact time of drug with the cornea, increasing the penetration through the complex anatomical structure of the eye, and providing controlled release of drugs into the eye tissues, which allows reducing the drug application frequency. The rest of the paper describes recommendedin vitroandin vivostudies to be performed for various ophthalmic drugs forms in order to assess whether the form is acceptable from the perspective of desired properties and patient’s compliance.

scholarly journals Consequences of exclusive expression in vivo of kit-ligand lacking the major proteolytic cleavage site

1998 ◽  
Vol 95 (20) ◽  
pp. 11903-11908 ◽  
Author(s):  
Y. Tajima ◽  
M. A. S. Moore ◽  
V. Soares ◽  
M. Ono ◽  
H. Kissel ◽  
...  
Keyword(s):  
Cleavage Site ◽  
Proteolytic Cleavage ◽  
Kit Ligand ◽  
Proteolytic Cleavage Site

Genetic analysis of oxygen defense mechanisms in Drosophila melanogaster and identification of a novel behavioural mutant with a Shaker phenotype

Genome ◽  
1996 ◽  
Vol 39 (4) ◽  
pp. 749-757 ◽  
Author(s):  
James M. Humphreys ◽  
Brenda Duyf ◽  
Mei-Ling A. Joiner ◽  
John P. Phillips ◽  
Arthur J. Hilliker
Keyword(s):  
Drosophila Melanogaster ◽  
Defense Mechanisms ◽  
Chromosome Region ◽  
Recessive Mutation ◽  
Ether Anaesthesia ◽  
Ros Metabolism ◽  
Chromosome Mutations ◽  
New Gene ◽  
New Alleles

Mutants of Drosophila melanogaster that lack Cu/Zn superoxide dismutase or urate are hypersensitive to reactive oxygen species (ROS) generated in vivo by the redox-cycling agent paraquat. We have subsequently employed paraquat as a selective agent to identify adult viable mutants potentially defective in other, perhaps unknown, components of ROS metabolism. Paraquat screening of ethyl methanesulfonate-induced second- and third-chromosome mutations yielded 24 paraquat hypersensitive mutants. Two mutants were identified as being new alleles of the previously identified doublesex (dsx) and pink (p) genes. The remainder of the mutations identified previously undescribed genes, including one second chromosome paraquat hypersensitive mutant that was found to exhibit shaking legs, abdomen pulsations, and body shuddering under ether anaesthesia. This recessive mutation was mapped to the polytene chromosome region of 48A5–48B2 and defines a new gene we named quiver (qvr). This mutation is similar in phenotype to the Shaker (Sh), ether-a-gogo (eag), and Hyperkinetic (Hk) mutations, all of which affect potassium channel function in D. melanogaster. Key words : Drosophila, paraquat, EMS-mutagenesis, Shaker, oxidative-stress.

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